WO2017038709A1 - Surface-assisted laser desorption/ionization method, mass spectrometry method and mass spectrometry device - Google Patents

Surface-assisted laser desorption/ionization method, mass spectrometry method and mass spectrometry device Download PDF

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Publication number
WO2017038709A1
WO2017038709A1 PCT/JP2016/075049 JP2016075049W WO2017038709A1 WO 2017038709 A1 WO2017038709 A1 WO 2017038709A1 JP 2016075049 W JP2016075049 W JP 2016075049W WO 2017038709 A1 WO2017038709 A1 WO 2017038709A1
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WO
WIPO (PCT)
Prior art keywords
sample
substrate
sample support
laser
hole
Prior art date
Application number
PCT/JP2016/075049
Other languages
French (fr)
Japanese (ja)
Inventor
康秀 内藤
小谷　政弘
孝幸 大村
Original Assignee
浜松ホトニクス株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN201680002988.2A priority Critical patent/CN107076705B/en
Priority to JP2016574483A priority patent/JP6105182B1/en
Priority to EP19217802.8A priority patent/EP3654365B1/en
Priority to EP23189772.9A priority patent/EP4257966A3/en
Priority to EP16841741.8A priority patent/EP3214436B1/en
Priority to CN201911035872.0A priority patent/CN110736784B/en
Application filed by 浜松ホトニクス株式会社 filed Critical 浜松ホトニクス株式会社
Priority to US15/571,568 priority patent/US10224195B2/en
Publication of WO2017038709A1 publication Critical patent/WO2017038709A1/en
Priority to US16/238,250 priority patent/US10679835B2/en
Priority to US16/864,919 priority patent/US11170985B2/en
Priority to US17/465,173 priority patent/US11646187B2/en
Priority to US18/117,632 priority patent/US11961728B2/en

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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2806—Means for preparing replicas of specimens, e.g. for microscopal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0004—Imaging particle spectrometry
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/26—Mass spectrometers or separator tubes

Definitions

  • the present invention relates to a surface-assisted laser desorption ionization method, a mass spectrometry method, and a mass spectrometer.
  • MALDI Matrix-Assisted Laser Desorption / Ionization
  • MALDI is known as a technique for ionizing a sample such as a biological sample for performing mass spectrometry or the like.
  • MALDI is a method of ionizing a sample by mixing a low molecular weight organic compound called a matrix that absorbs an ultraviolet laser with the sample and irradiating the sample with a laser.
  • a matrix that absorbs an ultraviolet laser with the sample and irradiating the sample with a laser.
  • soft ionization a high molecular weight substance
  • SALDI Surface-Assisted Laser Desorption / Ionization
  • a sample ionization method using SALDI there is a method in which anodized porous alumina, anodized porous silicon or the like having fine concave portions on the surface is used as a sample holding surface (see Patent Documents 1 and 2 below).
  • a sample to be analyzed is dropped on a sample holding surface having a fine recess, and the sample is ionized by irradiating a laser after drying.
  • the sample is displaced with respect to the substrate when the sample is dropped, so that the sample is ionized while maintaining the original sample position information (two-dimensional distribution of molecules constituting the sample). It is difficult. For this reason, it is difficult to use the above ionization method for imaging mass spectrometry or the like that measures what kind of molecules exist in each position of the sample region and images a two-dimensional distribution map of the sample molecules. . Further, even if a method of transferring the sample to the substrate instead of dropping the sample on the substrate is employed, there is a problem that the sample is displaced with respect to the substrate at the time of transferring the sample or uneven transfer of the sample occurs. .
  • an object of one aspect of the present invention is to provide a surface-assisted laser desorption / ionization method, a mass spectrometry method, and a mass spectrometer that can ionize a sample while maintaining the position information of the sample.
  • a surface-assisted laser desorption / ionization method includes a substrate provided with a plurality of through holes penetrating from one surface to the other surface, and a conductive layer made of a conductive material and covering at least one surface.
  • a first step in which a sample support is prepared a second step in which the sample is placed on the sample stage and the sample support is placed on the sample so that the other surface faces the sample, and a laser is provided on one side. Irradiating the sample, the third step of ionizing the sample that has moved from the other surface side to the one surface side through the through hole by capillary action.
  • a substrate provided with a plurality of through holes is arranged on the sample, so that it is directed from the other surface side of the substrate to the one surface side through the through holes by capillary action.
  • the sample can be raised. Thereby, the sample can be moved from the other surface side of the substrate to the one surface side while maintaining the position information of the sample (two-dimensional distribution of molecules constituting the sample).
  • the laser is irradiated on one surface of the substrate, and energy is transmitted to the sample moved to the one surface side through the conductive layer, whereby the sample is ionized.
  • the sample can be ionized while maintaining the position information of the sample. Therefore, according to the above method, the sample can be ionized while maintaining the position information of the sample by a simple operation of placing a substrate provided with a plurality of through holes on the sample.
  • the substrate may be formed by anodizing valve metal or silicon.
  • a sample support including a substrate provided with a plurality of through holes by anodizing a valve metal or silicon it is possible to appropriately realize the movement of the sample by the capillary phenomenon described above.
  • the width of the through hole may be 1 to 700 nm.
  • the thickness of the substrate may be 5 to 10 ⁇ m.
  • the sample support may further include a frame attached to the outer edge of one surface of the substrate.
  • the frame body suppresses bending of the substrate and facilitates handling when the sample support is supported or moved. As a result, it is possible to easily arrange the sample support on the sample in the second step.
  • the sample support may be fixed to the sample stage.
  • the sample and the sample support can be brought into close contact with each other, and the sample can be moved more smoothly by capillary action.
  • the side support of the sample support disposed on the sample is prevented, and the sample position shift due to the side slip of the sample support can be suppressed.
  • a sample support including a substrate made of a conductive material and provided with a plurality of through holes penetrating from one surface to the other surface is prepared.
  • a second step in which the sample is placed on the sample stage and the sample support is placed on the sample so that the other surface is in contact with the sample;
  • a third step in which the sample moved from the other surface side to the one surface side through the through hole is ionized.
  • the conductive layer can be omitted by using a substrate made of a conductive material, and the same effect as in the case of using the sample support including the conductive layer described above can be obtained.
  • a mass spectrometry method includes each step of the surface-assisted laser desorption / ionization method and a fourth step in which a sample ionized in the third step is detected. Laser irradiation and detection of the ionized sample in the fourth step are performed for each irradiation position while changing the laser irradiation position.
  • the sample can be ionized while maintaining the position information of the sample by a simple operation of placing the sample support on the sample.
  • the mass spectrometry method imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
  • a mass spectrometer includes a sample stage on which a sample is placed, a substrate provided with a plurality of through holes penetrating from one surface to the other surface, and a conductive material, and covers at least one surface.
  • a detection unit that detects a sample ionized by laser irradiation for each irradiation position.
  • the sample can be ionized while maintaining the position information of the sample by a simple operation of placing the sample support on the sample.
  • the laser irradiation unit irradiates the laser while changing the irradiation position, and the detection unit detects the ionized sample for each irradiation position, whereby the two-dimensional distribution of the sample molecules can be grasped. Therefore, according to the mass spectrometer, imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
  • a mass spectrometer is a sample support including a sample stage on which a sample is placed, and a substrate made of a conductive material and provided with a plurality of through holes penetrating from one surface to the other surface.
  • it is ionized by laser irradiation with a laser irradiation unit that irradiates a laser on one surface while changing the irradiation position while being arranged on the sample placed on the sample stage so that the other surface is in contact with the sample.
  • a detection unit that detects the sample for each irradiation position.
  • the conductive layer can be omitted by using a substrate made of a conductive material, and the same effect as in the case of using the sample support including the conductive layer described above can be obtained.
  • the present invention it is possible to provide a surface-assisted laser desorption / ionization method, a mass spectrometry method, and a mass spectrometer that can ionize a sample while maintaining the position information of the sample.
  • FIG. 3 is a cross-sectional view taken along line III-III in FIG.
  • FIG. 3 is an enlarged plan view in an effective region R of the sample support in FIG. 2.
  • FIG. 3 is a diagram showing a manufacturing process of the substrate of FIG.
  • It is a figure which shows the procedure of the mass spectrometry method which concerns on this embodiment.
  • It is a figure which shows the procedure of the mass spectrometry method which concerns on this embodiment.
  • the mass spectrometry method (including the surface-assisted laser desorption / ionization method) according to the present embodiment will be described with reference to FIG.
  • FIG. 1A in the mass spectrometry method, first, one sample 10 to be subjected to mass spectrometry is placed on the sample stage 1. Further, a sample support 2 including a substrate provided with a plurality of through holes is disposed on the sample 10.
  • the sample 10 to be analyzed is a thin-film biological sample (hydrated sample) such as a tissue slice.
  • the sample 10 moves from the lower surface side of the sample support 2 to the upper surface side of the sample support 2 through the through-hole by capillary action. And the sample 10 will be in the state which stays on the upper surface side of the sample support body 2 by surface tension.
  • the sample 10 moved to the upper surface side of the sample support 2 is irradiated with an ultraviolet laser on the upper surface side of the sample support 2, and is ionized in vacuum.
  • an ultraviolet laser on the upper surface side of the sample support 2
  • the sample 10 that has acquired energy becomes sample ions (ionized sample) 11 by being vaporized and acquiring electric charge.
  • the sample ions 11 thus released into the air are detected by the detector 3, and the detected sample ions 11 are measured. In this way, mass analysis of the sample 10 is performed.
  • the mass spectrometry method uses time-of-flight mass spectrometry (TOF-MS).
  • TOF-MS time-of-flight mass spectrometry
  • a ground electrode (not shown) is provided between the sample support 2 and the detector 3, and a predetermined voltage is applied to the sample support 2.
  • a potential difference is generated between the sample support 2 and the ground electrode, and the sample ions 11 generated on the upper surface side of the sample support 2 move while being accelerated toward the ground electrode by the potential difference.
  • the sample ion 11 flies in a drift space (Drift Space) provided between the ground electrode and the detector 3 where no electric and magnetic fields exist, and finally reaches the detector 3.
  • Drift Space drift space
  • FIG. 2 is a perspective view showing the appearance (substrate 21 and frame 22) of the sample support 2.
  • the substrate 21 is provided with a plurality of through-holes S
  • the sample support 2 includes an adhesive layer G for bonding the substrate 21 and the frame 22, and the surface of the substrate 21 and the frame 22 ( And a conductive layer 23 covering the inner surface of the through hole S).
  • FIG. 3 which is a cross-sectional view taken along line III-III in FIG. 2, in order to explain the arrangement configuration of the through hole S, the conductive layer 23, and the adhesive layer G, the through hole S, the conductive layer 23,
  • the dimensions of the adhesive layer G are shown larger than the actual dimensions.
  • the sample support 2 is a sample support for surface-assisted laser desorption / ionization (SALDI) from one surface 21a to the other surface 21b. It has a rectangular plate-like substrate 21 provided with a plurality of through-holes S, and a frame body 22 attached to an outer edge portion of one surface 21a of the substrate 21.
  • SALDI surface-assisted laser desorption / ionization
  • the shape of the one surface 21a and the other surface 21b of the substrate 21 is, for example, a square having a side length D1 of 1 cm.
  • the thickness d1 from the one surface 21a to the other surface 21b of the substrate 21 is 1 to 50 ⁇ m.
  • the substrate 21 is made of an insulating material.
  • the substrate 21 is an alumina porous film in which a plurality of through holes S having a substantially constant hole diameter are formed by, for example, anodizing Al (aluminum).
  • the substrate 21 is made of Ta (tantalum), Nb (niobium), Ti (titanium), Hf (hafnium), Zr (zirconium), Zn (zinc), W (tungsten), Bi (bismuth), Sb (antimony).
  • Ta tantalum
  • Nb niobium
  • Ti titanium
  • Hf hafnium
  • Zr zirconium
  • Zn zinc
  • W tungsten
  • Bi bismuth
  • Sb antimony
  • it may be formed by anodizing a valve metal other than Al, or by anodizing Si (silicon).
  • the frame body 22 is provided in a square ring shape along the outer edge portion of the one surface 21 a of the substrate 21.
  • the width D2 of the frame body 22 is 2 mm, for example.
  • the thickness d2 of the frame body 22 is, for example, 10 to 500 ⁇ m.
  • the effective area R of the one surface 21a of the substrate 21 that is not covered by the frame 22 is a square area of 0.6 mm square.
  • the effective region R functions as a region for moving the sample 10 from the other surface 21b to the one surface 21a by capillary action described later.
  • the frame body 22 is provided in a quadrangular annular shape, but may be provided in an annular shape along the outer edge portion of the substrate 21. By providing the frame body 22 in an annular shape, the bending of the substrate 21 is further suppressed as compared with the case where the frame body 22 is provided in a square annular shape.
  • the frame body 22 is bonded to the surface (one surface 21a) of the substrate 21 through the adhesive layer G.
  • a material for the adhesive layer G for example, an adhesive material with a low emission gas such as a low melting glass or a vacuum adhesive can be used.
  • the frame body 22 is bonded to the substrate 21 so as to overlap a portion where the through hole S is provided on the one surface 21 a of the substrate 21. For this reason, bending of the boundary surface between the portion of the substrate 21 where the frame body 22 is provided and the portion where the frame body 22 is not provided is allowed by the through hole S. Thereby, it is suppressed that the board
  • the frame body 22 has a thermal expansion coefficient substantially equal to that of the substrate 21.
  • the frame 22 is, for example, a ceramic member having the same composition as the substrate 21.
  • the frame 22 is, for example, glass or metal. In this manner, by making the thermal expansion coefficients of the substrate 21 and the frame body 22 close to each other, deformation (for example, distortion of the substrate 21 and the frame body 22 during thermal expansion) and the like due to a temperature change can be prevented.
  • the sample support 2 includes a conductive layer 23 that covers one surface 21 a, the other surface 21 b, the inner surface of the through-hole S, and the surface of the frame body 22.
  • the conductive layer 23 is a layer made of a conductive material provided to impart conductivity to the insulating substrate 21. However, even when the substrate 21 is made of a conductive material, the provision of the conductive layer 23 is not hindered.
  • a metal having low affinity (reactivity) with the sample 10 and high conductivity is preferable for the following reason.
  • the conductive layer 23 is formed of a metal such as Cu (copper) having a high affinity with the sample 10 such as protein
  • the sample 10 is in a state in which Cu atoms are attached to the sample molecule in the ionization process of the sample 10 described later. May ionize. That is, when the molecular weight of the sample ions 11 detected by the detector 3 is measured, the molecular weight of the attached sample is deviated from the actual molecular weight of the sample 10, so that accurate measurement cannot be performed. Therefore, the material of the conductive layer 23 is preferably a metal having a low affinity with the sample 10.
  • a highly conductive metal can apply a constant voltage more easily and stably. For this reason, by using a highly conductive metal as the conductive layer 23, it is easy to apply a constant voltage to the substrate 21 in order to generate a constant potential difference between the ground electrode and the substrate 21 described above. Further, since the metal having higher conductivity tends to have higher thermal conductivity, the energy of the laser irradiated on the substrate 21 can be efficiently transmitted to the sample 10 through the conductive layer 23. Therefore, the material of the conductive layer 23 is preferably a highly conductive metal.
  • the conductive layer 23 is made of Au or Pt by using a plating method, an atomic layer deposition (ALD) method, a vapor deposition method, a sputtering method, or the like on one surface 21a, the other surface 21b, and the through hole of the substrate 21. It can be formed by forming a film on the inner surface of S and the surface of the frame body 22.
  • ALD atomic layer deposition
  • Cr chromium
  • Ni nickel
  • Ti titanium
  • FIG. 4 is an enlarged plan view of the effective region R of the sample support 2.
  • a black part shows the through-hole S
  • a white part shows the partition part in which the through-hole S is not formed.
  • a plurality of through holes S having a substantially constant size are formed on the surface of the substrate 21.
  • the plurality of through-holes S only need to be formed in a size that allows movement (rise) from the other surface 21b of the sample 10 to the one surface 21a by capillary action described later.
  • the sizes of the through holes S may be uneven, or there may be portions where the plurality of through holes S are connected to each other.
  • the aperture ratio of the through-hole S in the effective region R (the area of the portion where the through-hole S is formed / the total area) is practically 10 to 80%, particularly preferably 60 to 80%.
  • the through-hole S extends from the one surface 21a side of the substrate 21 to the other surface 21b side.
  • the width d3 of the through hole S is 1 to 700 nm.
  • the thickness d4 of the conductive layer 23 is, for example, about 1 to 25 nm.
  • the width d3 of the through hole S is a hole width after the conductive layer 23 is formed in the through hole S.
  • FIG. 6A an Al substrate 50 as a material for the substrate 21 is prepared.
  • FIG. 6B by anodizing the Al substrate 50, the Al substrate 50 is oxidized from the surface, and an anodized film 51 having a plurality of recesses 51a is formed.
  • FIG. 6C the anodic oxide film 51 is peeled from the Al substrate 50, and the bottom 51b of the peeled anodic oxide film 51 is removed or subjected to a penetration treatment, so that the one surface 21a is changed to the other surface.
  • a substrate 21 provided with a plurality of through holes S penetrating through 21b is obtained.
  • the frame body 22 is attached to the outer edge portion of the substrate 21 via an adhesive layer G such as low melting point glass or a vacuum adhesive. Thereby, it will be in the state before the conductive layer 23 is formed among the sample support bodies 2 shown in FIG.
  • a conductive layer 23 made of Au or Pt is provided so as to cover one surface 21a, the other surface 21b, the inner surface of the through hole S, and the surface of the frame body 22 of the substrate 21.
  • the conductive layer 23 is formed by depositing Au or Pt on the one surface 21a, the other surface 21b, the inner surface of the through hole S, and the surface of the frame body 22 by a plating method, an atomic layer deposition method, or the like. Formed by.
  • the sample support 2 shown in FIG. 3 is manufactured.
  • the thickness d1 of the substrate 21 is adjusted to 1 to 50 ⁇ m, and the width d3 of the through hole S is adjusted to 1 to 700 nm.
  • the thickness d1 of the substrate 21 and the width d3 of the through hole S are determined in advance by appropriately setting the conditions such as the thickness of the Al substrate 50 prepared first and the temperature and voltage in anodization. It is formed in a size (a size included in the above range).
  • the mass spectrometer 100 includes a sample stage 1 on which a sample 10 is placed, a laser irradiation unit 4, and a detector (detection unit) 3.
  • the laser irradiation unit 4 irradiates the surface 21 a with the laser L while changing the irradiation position in a state where the sample support 2 is arranged on the sample 10 placed on the sample stage 1.
  • the sample support 2 is placed on the sample 10 such that the other surface 21 b comes into contact with the sample 10 via the conductive layer 23.
  • the laser L irradiated by the laser irradiation unit 4 is an ultraviolet laser such as a nitrogen laser (N2 laser) having a wavelength of 337 nm, for example.
  • the detector 3 detects the sample 10 (sample ion 11) ionized by the laser L irradiation by the laser irradiation unit 4 for each irradiation position. Specifically, the laser irradiation unit 4 scans the effective region R of the sample support 2 two-dimensionally according to a predetermined movement width and movement direction, and irradiates the laser L at each scanning position. The detector 3 detects sample ions 11 generated by irradiation with the laser L at each scanning position. Thereby, it becomes possible to perform mass spectrometry for every position on the effective region R. By integrating the mass analysis results at each position in the sample 10 obtained in this way, it is possible to perform imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules.
  • the mass analysis procedure by the mass spectrometer 100 will be described in detail with reference to FIGS.
  • the sample support 2 described above is prepared (first step).
  • the sample support 2 may be prepared by a person who performs mass spectrometry using the mass spectrometer 100, or may be prepared by obtaining from the manufacturer and seller of the sample support 2. May be.
  • the sample 10 to be subjected to mass spectrometry is placed on the placement surface 1a of the sample stage 1, and as shown in FIG.
  • the sample support 2 is disposed on the sample 10 so that 21b comes into contact with the sample 10 through the conductive layer 23 (see FIG. 3) (second step).
  • the sample support 2 is placed on the sample 10 so that the sample 10 is included in the effective region R in plan view. Placed in.
  • a solution for lowering the viscosity of the sample 10 may be mixed with the sample 10 in order to make the movement of the sample 10 due to capillary action, which will be described later, smooth.
  • the sample support 2 is fixed to the sample stage 1 (continuation of the second step).
  • the four sides of the sample support 2 (the upper surface and the side surfaces of the frame body 22 and the side surfaces of the substrate 21) are placed on the mounting surface 1a of the sample table 1 by an adhesive tape T having conductivity such as a carbon tape. It is fixed against.
  • the sample support 2 by fixing the sample support 2 to the sample stage 1, the sample 10 and the sample support 2 can be brought into close contact with each other, and the sample 10 can be moved more smoothly by capillary action described later.
  • the skidding of the sample support 2 arranged on the sample 10 is prevented, and it is possible to prevent the position information of the sample 10 from being lost due to the skidding of the sample support 2.
  • the sample stage 1 and the sample support 2 are electrically connected by the adhesive tape T having conductivity. Therefore, a predetermined voltage is applied to the substrate 21 by applying a predetermined current to the sample stage 1 in a state where the sample support 2 is fixed to the sample stage 1 with the adhesive tape T as shown in FIG. be able to. Thereby, a constant potential difference can be generated between the ground electrode and the substrate 21 described above.
  • the conductive layer 23 covers the frame body 22 and the adhesive tape T is in contact with the conductive layer 23 on the frame body 22, the sample support 2 and the power source (the current on the sample stage 1). Contact with a predetermined power source) can be taken on the frame 22. That is, it is possible to make contact between the sample support 2 and the power source without reducing the effective area R on the substrate 21.
  • the sample 10 passes through the through-hole S from the other surface 21b side of the substrate 21 by capillary action. And move (rise) toward the surface 21a. And the sample 10 will be in the state which stays on the one surface 21a side of the sample support body 2 by surface tension.
  • the mounting surface 1a of the sample stage 1 and the one surface 21a and the other surface 21b of the substrate 21 are arranged substantially parallel to each other. Therefore, the sample 10 placed on the sample table 1 is formed on the one surface 21a through the through hole S from the other surface 21b side of the substrate 21 along the direction orthogonal to the placement surface 1a of the sample table 1 by capillary action. Will move to the side.
  • the position information of the sample 10 (each sample molecule constituting the sample 10) is maintained before and after the movement due to the capillary phenomenon.
  • the two-dimensional coordinates (positions in a two-dimensional plane parallel to the mounting surface 1a of the sample table 1) of each sample molecule constituting the sample 10 do not change greatly before and after the movement due to the capillary phenomenon. Therefore, the capillary 10 can move the sample 10 from the other surface 21b side of the substrate 21 to the one surface 21a side while maintaining the position information of the sample 10.
  • the laser irradiation unit 4 irradiates the surface 21a of the substrate 21 with the laser L, and the sample 10 moved from the other surface 21b side to the one surface 21a side through the through hole S due to capillary action. Is ionized (third step). Then, the ionized sample 10 (sample ion 11) is detected by the detector 3 (fourth step). The irradiation of the laser L in the third step and the detection of the sample ions 11 in the fourth step are performed for each irradiation position while changing the irradiation position of the laser L.
  • the laser irradiation unit 4 scans the effective region R according to a predetermined movement width and movement direction, and irradiates the laser L at each irradiation position while changing the irradiation position of the laser L.
  • the detector 3 detects the sample ion 11 discharge
  • the substrate 21 provided with a plurality of through-holes S is disposed on the sample 10, so that the other of the substrate 21 is caused by capillary action.
  • the sample 10 can be raised from the surface 21b side through the through hole S toward the one surface 21a side. Thereby, the sample 10 can be moved from the other surface 21b side of the substrate 21 to the one surface 21a side while maintaining the position information of the sample 10 (two-dimensional distribution of molecules constituting the sample 10).
  • the laser L is irradiated on the one surface 21 a of the substrate 21, and energy is transmitted to the sample 10 moved to the one surface 21 a side through the conductive layer 23, whereby the sample 10 is ionized.
  • the sample 10 can be ionized while maintaining the position information of the sample 10. Therefore, according to the above method, the sample 10 can be ionized while maintaining the position information of the sample 10 by a simple operation of placing the substrate 21 provided with the plurality of through holes S on the sample 10. .
  • the sample support 2 including the substrate 21 provided with a plurality of through holes S by anodizing Al by using the sample support 2 including the substrate 21 provided with a plurality of through holes S by anodizing Al, the movement of the sample 10 by the capillary phenomenon described above can be appropriately realized.
  • the same effect can be obtained by using the sample support 2 provided with the substrate 21 obtained by anodizing a valve metal other than Al or Si instead of Al.
  • the sample 10 can be moved more smoothly by the capillary phenomenon described above.
  • the sample support 2 includes the frame 22 attached to the outer edge portion of the one surface 21a of the substrate 21, the frame 22 suppresses the bending of the substrate 21 and supports the sample support 2. Handling when moving or moving. For this reason, arrangement
  • the sample 10 is ionized while maintaining the position information of the sample 10 by a simple operation of placing the sample support 2 on the sample 10. Can do. Then, by detecting the ionized sample 10 (sample ions 11) for each irradiation position while changing the irradiation position of the laser L, the two-dimensional distribution of the sample molecules can be grasped. Therefore, according to the mass spectrometry method, imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
  • the sample 10 can be ionized while maintaining the position information of the sample 10 by a simple operation of placing the sample support 2 on the sample 10. Then, the laser irradiation unit 4 irradiates the laser L while changing the irradiation position, and the detector 3 detects the ionized sample 10 (sample ion 11) for each irradiation position, thereby obtaining a two-dimensional distribution of the sample molecules. I can grasp it. Therefore, according to the mass spectrometer 100, imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
  • the substrate 21 may be made of a conductive material such as a semiconductor.
  • the sample support 2 can omit the conductive layer 23 for imparting conductivity to the substrate 21.
  • the sample support 2 does not include the conductive layer 23
  • the sample support 2 is arranged on the sample 10 so that the other surface 21b is in direct contact with the sample 10 in the second step.
  • the same effect as in the case of using the sample support 2 including the conductive layer 23 described above can be obtained. Can do.
  • the ionization of the sample 10 by the surface-assisted laser desorption ionization method is not limited to the imaging mass spectrometry of the sample 10 described in the present embodiment, but other measurements such as ion mobility measurement. It can also be used for experiments.
  • the conductive layer 23 may be provided by vapor deposition or the like so as to cover at least the one surface 21a of the substrate 21. That is, the conductive layer 23 may not be provided on the other surface 21b of the substrate 21 and the inner surface of the through hole S.
  • the sample support 2 is disposed on the sample 10 so that the other surface 21b faces the sample 10, and the other surface 21b directly contacts the sample 10.
  • the conductive layer 23 is provided so as to cover at least one surface 21 a of the substrate 21 and the surface of the frame body 22, contact between the substrate 21 and the electrode can be made on the frame body 22.
  • 10 to 13 show the relationship between the hole width of the through hole S and the mass spectrum measured by the mass spectrometry method.
  • the conductive layer 23 here, Pt
  • the conductive layer 23 is not provided on the other surface 21b of the substrate 21 and the inner surface of the through-hole S, but is provided so as to cover the surface 21a and the surface of the frame body 22.
  • the thickness d1 of the substrate 21 is 10 ⁇ m
  • 10 to 13 (a) is a measurement result when the hole width of the through hole S is 50 nm, and (b) is a measurement result when the hole width of the through hole S is 100 nm.
  • c) is a measurement result when the hole width of the through-hole S is 200 nm
  • (d) is a measurement result when the hole width of the through-hole S is 300 nm
  • (e) is a hole of the through-hole S.
  • (F) is a measurement result when the hole width of the through-hole S is 500 nm
  • (g) is a measurement result when the hole width of the through-hole S is 600 nm. It is a result and (h) is a measurement result when the hole width of the through-hole S is 700 nm.
  • the vertical axis represents the signal intensity (Intensity) normalized with the peak value set to 100 (%).
  • FIG. 14 shows the relationship between the thickness d1 (Thickness) of the substrate 21 and the signal intensity of the peak measured by the mass spectrometry method.
  • the vertical axis represents the relative signal intensity when the signal intensity when the thickness d1 of the substrate 21 is 10 ⁇ m is “1”.
  • the conductive layer 23 here, Pt
  • the hole width of the through hole S is 200 nm.
  • the signal intensity when the thickness d1 of the substrate 21 was 10 ⁇ m was sufficiently large in mass spectrometry. Further, as shown in FIG. 14, the signal strength tends to increase as the thickness d1 of the substrate 21 decreases, and sufficient signal strength is obtained when the thickness d1 of the substrate 21 is in the range of 3 to 10 ⁇ m. It was. On the other hand, from the viewpoint of ensuring the substrate strength, the thickness d1 of the substrate 21 is preferably large. Therefore, the thickness d1 of the substrate 21 may be 5 to 10 ⁇ m. Thereby, while maintaining the intensity
  • the said embodiment demonstrated the form which fixes the frame 22 of the sample support body 2 to the sample stand 1 with the adhesive tape T
  • the form which fixes the sample support body 2 to the sample stand 1 is not limited to the said form.
  • variations of the embodiment in which the sample support 2 is fixed to the sample stage 1 will be described together with the first to third modifications of the sample support 2 with reference to FIGS. 15 to 17, illustration of the conductive layer 23 and the through hole S is omitted. 16 and 17, the illustration of the adhesive layer G that bonds the frame and the substrate is also omitted.
  • the sample support 2 ⁇ / b> A As shown in FIG. 15, the sample support 2 ⁇ / b> A according to the first modification has a frame 22 not provided on the substrate 21, and the adhesive tape T is directly attached to one surface 21 a of the substrate 21. Thus, it is mainly different from the sample support 2.
  • the adhesive tape T is affixed to the outer edge portion of the one surface 21 a so that the adhesive surface Ta faces the one surface 21 a of the substrate 21 and has a portion extending outward from the outer edge of the substrate 21.
  • the adhesive surface Ta can be attached to the outer edge of the substrate 21 and the mounting surface 1 a of the sample stage 1.
  • the sample support 2 ⁇ / b> A is fixed to the sample table 1 by the adhesive tape T. According to the sample support 2A, the followability of the substrate 21 with respect to the sample 10 can be improved, for example, when performing mass analysis of the sample 10 having an uneven surface.
  • the sample stage 1 and the sample support 2A (specifically, the conductive layer 23 provided on the one surface 21a of the substrate 21) have conductivity. It is electrically connected via an adhesive tape T. Accordingly, as shown in FIG. 15, a predetermined voltage is applied to the substrate 21 by applying a predetermined current to the sample stage 1 with the sample support 2 fixed to the sample stage 1 via the adhesive tape T. it can.
  • the sample support 2 ⁇ / b> A is distributed in a state where the adhesive tape T is attached to the outer edge of the substrate 21, and the adhesive protective sheet is provided on the adhesive surface Ta of the portion extending outside the outer edge of the substrate 21. May be allowed.
  • the user of the sample support 2A peels off the adhesive protective sheet immediately before fixing the sample support 2A to the sample stage 1, and attaches the adhesive surface Ta to the mounting surface 1a, whereby mass analysis of the sample 10 is performed. Can be easily prepared.
  • the sample support 2 ⁇ / b> B according to the second modification is mainly different from the sample support 2 in that it includes a frame body 122 having a portion extending outside the outer edge of the substrate 21. To do. Such a frame body 122 can appropriately suppress breakage of the end portion of the substrate 21 when carrying the sample support 2B. Further, as shown in FIG. 16, an insertion hole 122 a through which the screw 30 is inserted is provided in a portion of the frame body 122 that extends outside the outer edge of the substrate 21.
  • the sample support 2B can be securely fixed to the sample stage 1A by screwing.
  • the sample support 2B can be fixed to the sample stage 1A by inserting the screw 30 through the insertion hole 122a and the screw hole 1b.
  • the sample stage 1A and the sample support 2B (specifically, the conductive layer 23 formed on the surface of the frame body 122). Is electrically connected via a screw 30. Accordingly, a predetermined voltage can be applied to the substrate 21 by flowing a predetermined current through the sample stage 1A in a state where the sample support 2B is fixed to the sample stage 1A via the screw 30 as shown in FIG. .
  • the sample support 2C according to the third modification is provided on the outer edge portion of the other surface 21b of the substrate 21, and has an adhesive surface 24a facing in the direction from the one surface 21a to the other surface 21b.
  • the main difference from the sample support 2 is that the layer 24 is provided.
  • the adhesive layer 24 is, for example, a double-sided tape having a thickness set in advance according to the thickness of the sample 10 to be measured.
  • one adhesive surface 24 b of the adhesive layer 24 is previously attached to the outer edge portion of the other surface 21 b of the substrate 21, and the other adhesive surface 24 a of the adhesive layer 24 attaches the sample support 2 C to the sample stage 1.
  • fixing it is affixed on the mounting surface 1a.
  • the configuration for fixing the sample support 2C to the sample stage 1 can be simplified.
  • the sample stage 1 and the sample support 2C are interposed via the adhesive layer 24. Electrically connected. Accordingly, as shown in FIG. 17, a predetermined voltage is applied to the substrate 21 by flowing a predetermined current through the sample stage 1 with the sample support 2 ⁇ / b> C fixed to the sample stage 1 via the adhesive layer 24. it can.
  • the sample support 2C may be circulated with the adhesive surface 24b of the adhesive layer 24 attached to the outer edge portion of the other surface 21b of the substrate 21 and the adhesive protective sheet provided on the adhesive surface 24a. Good. In this case, the user of the sample support 2C peels off the adhesive protective sheet immediately before fixing the sample support 2C to the sample stage 1, and attaches the adhesive surface 24a to the mounting surface 1a, whereby mass analysis of the sample 10 is performed. Can be easily prepared.
  • the sample supports 2, 2A, 2B, and 2C may be baked after the conductive layer 23 is formed. That is, the manufacturing process of the sample support in the above embodiment may include a firing step of firing the sample support after the conductive layer 23 is formed.
  • a baking process is performed on the sample support including the substrate 21, the frame body 22, and the conductive layer 23.
  • a baking process is performed on the sample support including the substrate 21 and the conductive layer 23.
  • the crystallinity of the conductive layer 23 (for example, Pt) can be improved, and a sample support more suitable for mass spectrometry can be obtained.
  • the firing of the sample support is performed by conducting a conductive material (here, Pt) in the X-ray diffraction (XRD) measurement on the conductive layer 23 (sample support) after firing. It is preferably carried out so that a diffraction peak of the crystal of
  • “shows the diffraction peak of the crystal of the conductive material” means that the diffraction pattern (peak intensity, etc.) of the crystal of the conductive material is higher than the measurement result obtained by the X-ray diffraction measurement on the sample support before firing. Is clearly indicated.
  • FIG. 18 shows a mass spectrum measured by the mass spectrometer 100 having the sample support before firing.
  • FIG. 18B shows a mass spectrum measured by a mass spectrometer 100 having a sample support after firing at a firing temperature of 400 ° C. 18A and 18B, the measurement conditions other than the presence or absence of firing (the type of sample and the configuration of the sample support, etc.) are the same.
  • the vertical axes of FIGS. 18A and 18B indicate the peak signal intensity (that is, the peak value in the graph of FIG. 18B) when the sample support after firing is used as “100”. "Indicates the relative signal strength. As shown in FIG.
  • the signal intensity in mass spectrometry can be improved as compared with the case of using the sample support before firing. In this way, a sample support more suitable for mass spectrometry can be obtained by performing the above-described firing step.

Abstract

A surface-assisted laser desorption/ionization method according to one aspect of the present invention includes: a first step of preparing a sample supporting body 2 provided with a base plate 21 having a plurality of through-holes S which penetrate through from one surface 21a to another surface 21b, and an electrically conductive layer 23 covering at least the one surface 21a; a second step of placing a sample 10 on a sample stage 1 and disposing the sample supporting body 2 on the sample 10 in such a way that the other surface 21b opposes the sample 10; and a third step of irradiating the one surface 21a with a laser L to ionize the sample 10 which has migrated through the through-holes S by means of capillary action from the other surface 21b side to the one surface 21a side.

Description

表面支援レーザ脱離イオン化法、質量分析方法、及び質量分析装置Surface-assisted laser desorption / ionization method, mass spectrometry method, and mass spectrometer
　本発明は、表面支援レーザ脱離イオン化法、質量分析方法、及び質量分析装置に関する。 The present invention relates to a surface-assisted laser desorption ionization method, a mass spectrometry method, and a mass spectrometer.
　従来、質量分析等を行うために生体試料等の試料をイオン化する手法として、マトリックス支援レーザ脱離イオン化法（ＭＡＬＤＩ：Matrix-Assisted　Laser　Desorption/Ionization）が知られている。ＭＡＬＤＩは、紫外線レーザを吸収するマトリックスと呼ばれる低分子量の有機化合物を試料と混合し、これにレーザを照射することにより、試料をイオン化する手法である。この手法によれば、熱に不安定な物質や高分子量物質を非破壊でイオン化すること（いわゆるソフトイオン化）ができる。しかし、ＭＡＬＤＩでは、マトリックス由来のバックグラウンドノイズが発生する。 Conventionally, Matrix-Assisted Laser Desorption / Ionization (MALDI) is known as a technique for ionizing a sample such as a biological sample for performing mass spectrometry or the like. MALDI is a method of ionizing a sample by mixing a low molecular weight organic compound called a matrix that absorbs an ultraviolet laser with the sample and irradiating the sample with a laser. According to this technique, it is possible to non-destructively ionize a thermally unstable substance or a high molecular weight substance (so-called soft ionization). However, in MALDI, matrix-derived background noise occurs.
　そこで、マトリックスを使用せずにイオン化を行う手法として、表面に微細な凹凸構造を有する基板を使用することにより、試料をイオン化する表面支援レーザ脱離イオン化法（ＳＡＬＤＩ：Surface-Assisted　Laser　Desorption/Ionization）が知られている。例えば、ＳＡＬＤＩによる試料のイオン化法としては、表面に微細な凹部を有する陽極酸化ポーラスアルミナ及び陽極酸化ポーラスシリコン等を試料保持面として使用する方法が存在する（下記特許文献１及び２参照）。このイオン化法では、微細な凹部を有する試料保持面に対して分析対象の試料を滴下し、乾燥後にレーザを照射することにより、試料のイオン化を行っている。 Therefore, as a technique for performing ionization without using a matrix, a surface-assisted laser desorption / ionization method (SALDI: Surface-Assisted Laser Desorption / Ionization) that ionizes a sample by using a substrate having a fine uneven structure on the surface. )It has been known. For example, as a sample ionization method using SALDI, there is a method in which anodized porous alumina, anodized porous silicon or the like having fine concave portions on the surface is used as a sample holding surface (see Patent Documents 1 and 2 below). In this ionization method, a sample to be analyzed is dropped on a sample holding surface having a fine recess, and the sample is ionized by irradiating a laser after drying.
特許第５１２９６２８号公報Japanese Patent No. 5129628 米国特許６２８８３９０号公報US Pat. No. 6,288,390
　しかしながら、上述のイオン化法では、試料の滴下時に基板に対する試料の位置ずれが生じてしまうため、元々の試料の位置情報（試料を構成する分子の二次元分布）を維持したまま試料のイオン化を行うことは困難である。このため、試料領域の各位置にどのような分子がどれだけ存在するかを測定し、試料分子の二次元分布図を画像化するイメージング質量分析等に上記イオン化法を利用することは困難である。また、基板上に試料を滴下する代わりに試料を基板に転写させる方式を採用したとしても、試料の転写時に基板に対する試料の位置ずれが生じたり、試料の転写ムラが生じたりするといった問題がある。 However, in the ionization method described above, the sample is displaced with respect to the substrate when the sample is dropped, so that the sample is ionized while maintaining the original sample position information (two-dimensional distribution of molecules constituting the sample). It is difficult. For this reason, it is difficult to use the above ionization method for imaging mass spectrometry or the like that measures what kind of molecules exist in each position of the sample region and images a two-dimensional distribution map of the sample molecules. . Further, even if a method of transferring the sample to the substrate instead of dropping the sample on the substrate is employed, there is a problem that the sample is displaced with respect to the substrate at the time of transferring the sample or uneven transfer of the sample occurs. .
　そこで、本発明の一側面は、試料の位置情報を維持したまま試料のイオン化を行うことができる表面支援レーザ脱離イオン化法、質量分析方法、及び質量分析装置を提供することを目的とする。 Accordingly, an object of one aspect of the present invention is to provide a surface-assisted laser desorption / ionization method, a mass spectrometry method, and a mass spectrometer that can ionize a sample while maintaining the position information of the sample.
　本発明の一側面に係る表面支援レーザ脱離イオン化法は、一面から他面にかけて貫通する複数の貫通孔が設けられた基板と、導電性材料からなり、少なくとも一面を覆う導電層と、を備える試料支持体が用意される第１工程と、試料が試料台に載置され、且つ、他面が試料に対向するように試料支持体が試料上に配置される第２工程と、一面にレーザが照射されることにより、毛細管現象によって他面側から貫通孔を介して一面側に移動した試料がイオン化される第３工程と、を含む。 A surface-assisted laser desorption / ionization method according to one aspect of the present invention includes a substrate provided with a plurality of through holes penetrating from one surface to the other surface, and a conductive layer made of a conductive material and covering at least one surface. A first step in which a sample support is prepared, a second step in which the sample is placed on the sample stage and the sample support is placed on the sample so that the other surface faces the sample, and a laser is provided on one side. Irradiating the sample, the third step of ionizing the sample that has moved from the other surface side to the one surface side through the through hole by capillary action.
　上記表面支援レーザ脱離イオン化法によれば、複数の貫通孔が設けられた基板が試料上に配置されることで、毛細管現象によって、基板の他面側から貫通孔を介して一面側に向けて試料を上昇させることができる。これにより、試料の位置情報（試料を構成する分子の二次元分布）を維持したまま、試料を基板の他面側から一面側に移動させることができる。そして、レーザが基板の一面に照射され、一面側に移動した試料に導電層を介してエネルギーが伝達されることにより、試料がイオン化される。その結果、試料の位置情報を維持したまま試料のイオン化を行うことができる。従って、上記方法によれば、複数の貫通孔が設けられた基板を試料上に載置する簡単な操作によって、試料の位置情報を維持したまま試料のイオン化を行うことができる。 According to the surface-assisted laser desorption / ionization method, a substrate provided with a plurality of through holes is arranged on the sample, so that it is directed from the other surface side of the substrate to the one surface side through the through holes by capillary action. The sample can be raised. Thereby, the sample can be moved from the other surface side of the substrate to the one surface side while maintaining the position information of the sample (two-dimensional distribution of molecules constituting the sample). Then, the laser is irradiated on one surface of the substrate, and energy is transmitted to the sample moved to the one surface side through the conductive layer, whereby the sample is ionized. As a result, the sample can be ionized while maintaining the position information of the sample. Therefore, according to the above method, the sample can be ionized while maintaining the position information of the sample by a simple operation of placing a substrate provided with a plurality of through holes on the sample.
　基板はバルブ金属又はシリコンを陽極酸化することにより形成されていてもよい。バルブ金属又はシリコンを陽極酸化することで複数の貫通孔が設けられた基板を備える試料支持体を用いることにより、上述した毛細管現象による試料の移動を適切に実現することができる。 The substrate may be formed by anodizing valve metal or silicon. By using a sample support including a substrate provided with a plurality of through holes by anodizing a valve metal or silicon, it is possible to appropriately realize the movement of the sample by the capillary phenomenon described above.
　貫通孔の幅は１～７００ｎｍであってもよい。孔幅が１～７００ｎｍの貫通孔を有する基板を用いることにより、上述した毛細管現象による試料の移動をよりスムーズに行わせることができる。また、上記表面支援レーザ脱離イオン化法を用いた質量分析において十分な信号強度を得ることができる。 The width of the through hole may be 1 to 700 nm. By using a substrate having a through hole with a hole width of 1 to 700 nm, the sample can be moved more smoothly by the capillary phenomenon described above. In addition, sufficient signal intensity can be obtained in mass spectrometry using the surface-assisted laser desorption / ionization method.
　基板の厚さは５～１０μｍであってもよい。これにより、基板の強度を保つとともに、上記表面支援レーザ脱離イオン化法を用いた質量分析において十分な信号強度を得ることができる。 The thickness of the substrate may be 5 to 10 μm. Thereby, while maintaining the intensity | strength of a board | substrate, sufficient signal intensity | strength can be obtained in the mass spectrometry using the said surface assistance laser desorption ionization method.
　試料支持体が、基板の一面の外縁部に取り付けられた枠体を更に備えてもよい。枠体によって基板のしなりが抑制されるとともに、試料支持体を支持したり移動させたりする際の取り扱いが容易となる。その結果、第２工程における試料支持体の試料上への配置を容易に行うことができる。 The sample support may further include a frame attached to the outer edge of one surface of the substrate. The frame body suppresses bending of the substrate and facilitates handling when the sample support is supported or moved. As a result, it is possible to easily arrange the sample support on the sample in the second step.
　第２工程では、試料支持体が試料台に固定されてもよい。試料台に対して試料支持体を固定することにより、試料と試料支持体とを密着させ、毛細管現象による試料の移動をよりスムーズに行わせることができる。また、試料上に配置された試料支持体の横滑りが防止され、試料支持体の横滑りによる試料の位置ずれを抑制できる。 In the second step, the sample support may be fixed to the sample stage. By fixing the sample support to the sample stage, the sample and the sample support can be brought into close contact with each other, and the sample can be moved more smoothly by capillary action. In addition, the side support of the sample support disposed on the sample is prevented, and the sample position shift due to the side slip of the sample support can be suppressed.
　本発明の他の側面に係る表面支援レーザ脱離イオン化法は、導電性材料からなり、一面から他面にかけて貫通する複数の貫通孔が設けられた基板を備える試料支持体が用意される第１工程と、試料が試料台に載置され、且つ、他面が試料と接触するように試料支持体が試料上に配置される第２工程と、一面にレーザが照射されることにより、毛細管現象によって他面側から貫通孔を介して一面側に移動した試料がイオン化される第３工程と、を含む。 In the surface-assisted laser desorption / ionization method according to another aspect of the present invention, a sample support including a substrate made of a conductive material and provided with a plurality of through holes penetrating from one surface to the other surface is prepared. A second step in which the sample is placed on the sample stage and the sample support is placed on the sample so that the other surface is in contact with the sample; And a third step in which the sample moved from the other surface side to the one surface side through the through hole is ionized.
　上記表面支援レーザ脱離イオン化法では、導電性材料からなる基板を用いることにより導電層を省略できるとともに、上述した導電層を備える試料支持体を用いる場合と同様の効果を得ることができる。 In the surface-assisted laser desorption / ionization method, the conductive layer can be omitted by using a substrate made of a conductive material, and the same effect as in the case of using the sample support including the conductive layer described above can be obtained.
　本発明の一側面に係る質量分析方法は、上記表面支援レーザ脱離イオン化法の各工程と、第３工程においてイオン化された試料が検出される第４工程と、を含み、第３工程でのレーザの照射、及び第４工程でのイオン化された試料の検出は、レーザの照射位置を変えながら当該照射位置毎に行われる。 A mass spectrometry method according to one aspect of the present invention includes each step of the surface-assisted laser desorption / ionization method and a fourth step in which a sample ionized in the third step is detected. Laser irradiation and detection of the ionized sample in the fourth step are performed for each irradiation position while changing the laser irradiation position.
　上記質量分析方法によれば、試料上に試料支持体を配置する簡単な操作で、試料の位置情報を維持したまま試料のイオン化を行うことができる。そして、レーザの照射位置を変えながら、イオン化された試料を当該照射位置毎に検出することにより、試料分子の二次元分布を把握することができる。従って、上記質量分析方法によれば、簡単な操作で、試料分子の二次元分布図を画像化するイメージング質量分析を行うことができる。 According to the mass spectrometry method, the sample can be ionized while maintaining the position information of the sample by a simple operation of placing the sample support on the sample. By detecting the ionized sample for each irradiation position while changing the laser irradiation position, the two-dimensional distribution of the sample molecules can be grasped. Therefore, according to the mass spectrometry method, imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
　本発明の一側面に係る質量分析装置は、試料が載置される試料台と、一面から他面にかけて貫通する複数の貫通孔が設けられた基板と、導電性材料からなり、少なくとも一面を覆う導電層と、を備える試料支持体が、他面が試料に対向するように、試料台に載置された試料上に配置された状態で、照射位置を変えながら一面にレーザを照射するレーザ照射部と、レーザの照射によりイオン化された試料を照射位置毎に検出する検出部と、を備える。 A mass spectrometer according to one aspect of the present invention includes a sample stage on which a sample is placed, a substrate provided with a plurality of through holes penetrating from one surface to the other surface, and a conductive material, and covers at least one surface. Laser irradiation for irradiating one surface of the laser while changing the irradiation position in a state where the sample support including the conductive layer is placed on the sample placed on the sample stage so that the other surface faces the sample And a detection unit that detects a sample ionized by laser irradiation for each irradiation position.
　上記質量分析装置によれば、試料上に試料支持体を配置する簡単な操作で、試料の位置情報を維持したまま試料のイオン化を行うことができる。そして、レーザ照射部が照射位置を変えながらレーザを照射し、検出部がイオン化された試料を当該照射位置毎に検出することにより、試料分子の二次元分布を把握することができる。従って、上記質量分析装置によれば、簡単な操作で、試料分子の二次元分布図を画像化するイメージング質量分析を行うことができる。 According to the mass spectrometer, the sample can be ionized while maintaining the position information of the sample by a simple operation of placing the sample support on the sample. The laser irradiation unit irradiates the laser while changing the irradiation position, and the detection unit detects the ionized sample for each irradiation position, whereby the two-dimensional distribution of the sample molecules can be grasped. Therefore, according to the mass spectrometer, imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
　本発明の他の側面に係る質量分析装置は、試料が載置される試料台と、導電性材料からなり、一面から他面にかけて貫通する複数の貫通孔が設けられた基板を備える試料支持体が、他面が試料と接触するように、試料台に載置された試料上に配置された状態で、照射位置を変えながら一面にレーザを照射するレーザ照射部と、レーザの照射によりイオン化された試料を照射位置毎に検出する検出部と、を備える。 A mass spectrometer according to another aspect of the present invention is a sample support including a sample stage on which a sample is placed, and a substrate made of a conductive material and provided with a plurality of through holes penetrating from one surface to the other surface. However, it is ionized by laser irradiation with a laser irradiation unit that irradiates a laser on one surface while changing the irradiation position while being arranged on the sample placed on the sample stage so that the other surface is in contact with the sample. And a detection unit that detects the sample for each irradiation position.
　上記質量分析装置によれば、導電性材料からなる基板を用いることにより導電層を省略できるとともに、上述した導電層を備える試料支持体を用いる場合と同様の効果を得ることができる。 According to the mass spectrometer, the conductive layer can be omitted by using a substrate made of a conductive material, and the same effect as in the case of using the sample support including the conductive layer described above can be obtained.
　本発明によれば、試料の位置情報を維持したまま試料のイオン化を行うことができる表面支援レーザ脱離イオン化法、質量分析方法、及び質量分析装置を提供することができる。 According to the present invention, it is possible to provide a surface-assisted laser desorption / ionization method, a mass spectrometry method, and a mass spectrometer that can ionize a sample while maintaining the position information of the sample.
本発明の一実施形態に係る質量分析方法の概略を示す図である。It is a figure which shows the outline of the mass spectrometry method which concerns on one Embodiment of this invention. 本実施形態に係る質量分析方法に用いられる試料支持体の斜視図である。It is a perspective view of the sample support body used for the mass spectrometry method concerning this embodiment. 図２のＩＩＩ－ＩＩＩ線に沿った断面図である。FIG. 3 is a cross-sectional view taken along line III-III in FIG. 図２の試料支持体の実効領域Ｒにおける拡大平面図である。FIG. 3 is an enlarged plan view in an effective region R of the sample support in FIG. 2. 図２の試料支持体の要部拡大断面図である。It is a principal part expanded sectional view of the sample support body of FIG. 図２の基板の製造工程を示す図である。FIG. 3 is a diagram showing a manufacturing process of the substrate of FIG. 本実施形態に係る質量分析方法の手順を示す図である。It is a figure which shows the procedure of the mass spectrometry method which concerns on this embodiment. 本実施形態に係る質量分析方法の手順を示す図である。It is a figure which shows the procedure of the mass spectrometry method which concerns on this embodiment. 本実施形態に係る質量分析方法の手順を示す図である。It is a figure which shows the procedure of the mass spectrometry method which concerns on this embodiment. 貫通孔の孔幅とマススペクトルとの関係を示す図である。It is a figure which shows the relationship between the hole width of a through-hole, and a mass spectrum. 貫通孔の孔幅とマススペクトルとの関係を示す図である。It is a figure which shows the relationship between the hole width of a through-hole, and a mass spectrum. 貫通孔の孔幅とマススペクトルとの関係を示す図である。It is a figure which shows the relationship between the hole width of a through-hole, and a mass spectrum. 貫通孔の孔幅とマススペクトルとの関係を示す図である。It is a figure which shows the relationship between the hole width of a through-hole, and a mass spectrum. 基板の厚さと信号強度との関係を示す図である。It is a figure which shows the relationship between the thickness of a board | substrate, and signal strength. 試料支持体の第１の変形例を示す図である。It is a figure which shows the 1st modification of a sample support body. 試料支持体の第２の変形例を示す図である。It is a figure which shows the 2nd modification of a sample support body. 試料支持体の第３の変形例を示す図である。It is a figure which shows the 3rd modification of a sample support body. 焼成前の試料支持体を用いた質量分析によるマススペクトルと焼成後の試料支持体を用いた質量分析によるマススペクトルとを示す図である。It is a figure which shows the mass spectrum by mass spectrometry using the sample support body before baking, and the mass spectrum by mass spectrometry using the sample support body after baking.
　以下、図面を参照しながら、本発明の好適な実施形態について詳細に説明する。なお、各図において同一部分又は相当部分には同一符号を付し、重複する説明は省略する。また、図面に示される各部材（又は部位）の寸法又は寸法の比率は、説明をわかり易くするために、実際の寸法又は寸法の比率とは異なることがある。 Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. In addition, in each figure, the same code | symbol is attached | subjected to the same part or an equivalent part, and the overlapping description is abbreviate | omitted. In addition, the size or the ratio of dimensions of each member (or part) shown in the drawings may be different from the actual size or ratio of dimensions for easy understanding of the description.
　図１を用いて、本実施形態に係る質量分析方法（表面支援レーザ脱離イオン化法を含む）の概要について説明する。図１の（ａ）に示すように、上記質量分析方法では、まず、質量分析の対象となる一の試料１０が試料台１に載置される。さらに、複数の貫通孔が設けられた基板を備える試料支持体２が、試料１０の上に配置される。ここで、分析対象となる試料１０は、例えば組織切片等の薄膜状の生体試料（含水試料）である。 The outline of the mass spectrometry method (including the surface-assisted laser desorption / ionization method) according to the present embodiment will be described with reference to FIG. As shown in FIG. 1A, in the mass spectrometry method, first, one sample 10 to be subjected to mass spectrometry is placed on the sample stage 1. Further, a sample support 2 including a substrate provided with a plurality of through holes is disposed on the sample 10. Here, the sample 10 to be analyzed is a thin-film biological sample (hydrated sample) such as a tissue slice.
　続いて、図１の（ｂ）に示すように、試料１０は、毛細管現象によって、試料支持体２の下面側から貫通孔を介して試料支持体２の上面側に移動する。そして、試料１０は、試料支持体２の上面側に表面張力によって留まる状態となる。 Subsequently, as shown in FIG. 1B, the sample 10 moves from the lower surface side of the sample support 2 to the upper surface side of the sample support 2 through the through-hole by capillary action. And the sample 10 will be in the state which stays on the upper surface side of the sample support body 2 by surface tension.
　続いて、図１の（ｃ）に示すように、試料支持体２の上面側に紫外線レーザが照射されることにより、試料支持体２の上面側に移動した試料１０は、イオン化されて真空中に放出される。具体的には、紫外線レーザのエネルギーを吸収した試料支持体２から、試料支持体２の上面側に移動した試料１０にエネルギーが伝達される。そして、エネルギーを獲得した試料１０は、気化するとともに電荷を獲得することにより、試料イオン（イオン化された試料）１１となる。このようにして空気中に放出された試料イオン１１が検出器３によって検出され、検出された試料イオン１１の測定が行われる。このようにして、試料１０の質量分析が行われる。 Subsequently, as shown in FIG. 1C, the sample 10 moved to the upper surface side of the sample support 2 is irradiated with an ultraviolet laser on the upper surface side of the sample support 2, and is ionized in vacuum. To be released. Specifically, energy is transmitted from the sample support 2 that has absorbed the energy of the ultraviolet laser to the sample 10 that has moved to the upper surface side of the sample support 2. Then, the sample 10 that has acquired energy becomes sample ions (ionized sample) 11 by being vaporized and acquiring electric charge. The sample ions 11 thus released into the air are detected by the detector 3, and the detected sample ions 11 are measured. In this way, mass analysis of the sample 10 is performed.
　本実施形態に係る質量分析方法は、一例として、飛行時間型質量分析法（ＴＯＦ－ＭＳ：Time-of-Flight　Mass　Spectrometry）を利用する。ＴＯＦ－ＭＳの概要を以下に示す。ＴＯＦ－ＭＳでは、試料支持体２と検出器３との間に、グランド電極（不図示）が設けられるとともに、試料支持体２に対して所定の電圧が加えられる。これにより、試料支持体２とグランド電極との間に電位差が生じ、試料支持体２の上面側で発生した試料イオン１１は、当該電位差によってグランド電極に向かって加速しながら移動する。その後、試料イオン１１は、グランド電極から検出器３までの間に設けられた電場及び磁場が存在しないドリフト空間（Drift　Space）を飛行し、最終的に検出器３に到達する。ここで、試料支持体２とグランド電極との間の電位差はどの試料イオン１１に対しても一定であるため、各試料イオン１１に与えられるエネルギーは一定である。このため、分子量の小さい試料イオン１１ほどドリフト空間を高速で飛行し、短時間で検出器３に到達することになる。ＴＯＦ－ＭＳでは、このような試料イオン１１の検出器３への到達時間差に基づいて質量分析が行われる。 As an example, the mass spectrometry method according to the present embodiment uses time-of-flight mass spectrometry (TOF-MS). The outline of TOF-MS is shown below. In TOF-MS, a ground electrode (not shown) is provided between the sample support 2 and the detector 3, and a predetermined voltage is applied to the sample support 2. Thereby, a potential difference is generated between the sample support 2 and the ground electrode, and the sample ions 11 generated on the upper surface side of the sample support 2 move while being accelerated toward the ground electrode by the potential difference. Thereafter, the sample ion 11 flies in a drift space (Drift Space) provided between the ground electrode and the detector 3 where no electric and magnetic fields exist, and finally reaches the detector 3. Here, since the potential difference between the sample support 2 and the ground electrode is constant for any sample ion 11, the energy given to each sample ion 11 is constant. For this reason, the sample ion 11 having a smaller molecular weight flies in the drift space at a high speed and reaches the detector 3 in a short time. In TOF-MS, mass spectrometry is performed based on such a difference in arrival time of sample ions 11 to the detector 3.
　次に、図２～図５を用いて試料支持体２について説明する。図２は、試料支持体２の外観（基板２１及び枠体２２）を示す斜視図である。なお、実際には、基板２１には複数の貫通孔Ｓが設けられており、試料支持体２は、基板２１及び枠体２２を接着する接着層Ｇと、基板２１及び枠体２２の表面（貫通孔Ｓの内面を含む）を覆う導電層２３と、を備える。しかし、これらは基板２１及び枠体２２に対して非常に小さいため、図２においてはこれらの図示を省略している。一方、図２のＩＩＩ－ＩＩＩ線に沿った断面図である図３においては、貫通孔Ｓ、導電層２３、及び接着層Ｇの配置構成を説明するために、貫通孔Ｓ、導電層２３、及び接着層Ｇの寸法を実際の寸法よりも大きく図示している。 Next, the sample support 2 will be described with reference to FIGS. FIG. 2 is a perspective view showing the appearance (substrate 21 and frame 22) of the sample support 2. Actually, the substrate 21 is provided with a plurality of through-holes S, and the sample support 2 includes an adhesive layer G for bonding the substrate 21 and the frame 22, and the surface of the substrate 21 and the frame 22 ( And a conductive layer 23 covering the inner surface of the through hole S). However, since these are very small with respect to the board | substrate 21 and the frame 22, these illustration is abbreviate | omitted in FIG. On the other hand, in FIG. 3, which is a cross-sectional view taken along line III-III in FIG. 2, in order to explain the arrangement configuration of the through hole S, the conductive layer 23, and the adhesive layer G, the through hole S, the conductive layer 23, In addition, the dimensions of the adhesive layer G are shown larger than the actual dimensions.
　図２及び図３に示すように、試料支持体２は、表面支援レーザ脱離イオン化法（ＳＡＬＤＩ：Surface-Assisted　Laser　Desorption/Ionization）用の試料支持体であって、一面２１ａから他面２１ｂにかけて複数の貫通孔Ｓが設けられた矩形板状の基板２１と、基板２１の一面２１ａの外縁部に取り付けられた枠体２２と、を有する。 As shown in FIGS. 2 and 3, the sample support 2 is a sample support for surface-assisted laser desorption / ionization (SALDI) from one surface 21a to the other surface 21b. It has a rectangular plate-like substrate 21 provided with a plurality of through-holes S, and a frame body 22 attached to an outer edge portion of one surface 21a of the substrate 21.
　基板２１の一面２１ａ及び他面２１ｂの形状は、例えば一辺の長さＤ１が１ｃｍの正方形である。基板２１の一面２１ａから他面２１ｂまでの厚さｄ１は、１～５０μｍである。本実施形態では一例として、基板２１は、絶縁性材料からなっている。基板２１は、例えばＡｌ（アルミニウム）を陽極酸化することによって孔径がほぼ一定の貫通孔Ｓが複数形成されたアルミナポーラス皮膜である。なお、基板２１は、Ｔａ（タンタル）、Ｎｂ（ニオブ）、Ｔｉ（チタン）、Ｈｆ（ハフニウム）、Ｚｒ（ジルコニウム）、Ｚｎ（亜鉛）、Ｗ（タングステン）、Ｂｉ（ビスマス）、Ｓｂ（アンチモン）等のＡｌ以外のバルブ金属を陽極酸化することにより形成されてもよいし、Ｓｉ（シリコン）を陽極酸化することによって形成されてもよい。 The shape of the one surface 21a and the other surface 21b of the substrate 21 is, for example, a square having a side length D1 of 1 cm. The thickness d1 from the one surface 21a to the other surface 21b of the substrate 21 is 1 to 50 μm. In the present embodiment, as an example, the substrate 21 is made of an insulating material. The substrate 21 is an alumina porous film in which a plurality of through holes S having a substantially constant hole diameter are formed by, for example, anodizing Al (aluminum). The substrate 21 is made of Ta (tantalum), Nb (niobium), Ti (titanium), Hf (hafnium), Zr (zirconium), Zn (zinc), W (tungsten), Bi (bismuth), Sb (antimony). For example, it may be formed by anodizing a valve metal other than Al, or by anodizing Si (silicon).
　枠体２２は、基板２１の一面２１ａの外縁部に沿って四角環状に設けられている。枠体２２の幅Ｄ２は、例えば２ｍｍである。枠体２２の厚さｄ２は、例えば１０～５００μｍである。基板２１の一面２１ａのうち枠体２２に覆われていない実効領域Ｒは、０．６ｍｍ四方の正方形領域となっている。実効領域Ｒは、後述する毛細管現象によって試料１０を他面２１ｂから一面２１ａに移動させるための領域として機能する。枠体２２が基板２１の外縁部に設けられることにより、基板２１のしなりが抑制される。また、枠体２２が設けられた部分を固定したり把持したりすることが可能となるため、試料支持体２を支持したり移動させたりする際の取扱いが容易となる。なお、本実施形態では、枠体２２は四角環状に設けられているが、基板２１の外縁部に沿って円環状に設けられていてもよい。枠体２２を円環状に設けることで、枠体２２を四角環状に設けた場合よりも基板２１のしなりがより一層抑制される。 The frame body 22 is provided in a square ring shape along the outer edge portion of the one surface 21 a of the substrate 21. The width D2 of the frame body 22 is 2 mm, for example. The thickness d2 of the frame body 22 is, for example, 10 to 500 μm. The effective area R of the one surface 21a of the substrate 21 that is not covered by the frame 22 is a square area of 0.6 mm square. The effective region R functions as a region for moving the sample 10 from the other surface 21b to the one surface 21a by capillary action described later. By providing the frame 22 at the outer edge of the substrate 21, the bending of the substrate 21 is suppressed. Further, since the portion provided with the frame body 22 can be fixed or grasped, the handling when the sample support 2 is supported or moved is facilitated. In the present embodiment, the frame body 22 is provided in a quadrangular annular shape, but may be provided in an annular shape along the outer edge portion of the substrate 21. By providing the frame body 22 in an annular shape, the bending of the substrate 21 is further suppressed as compared with the case where the frame body 22 is provided in a square annular shape.
　図３に示すように、枠体２２は、接着層Ｇを介して基板２１の表面（一面２１ａ）に接着されている。接着層Ｇの材料としては、例えば低融点ガラスや真空用接着剤等の放出ガスの少ない接着材料を用いることができる。なお、本実施形態では一例として、枠体２２は、基板２１の一面２１ａにおいて貫通孔Ｓが設けられた部分と重なるようにして、基板２１に接着されている。このため、基板２１において枠体２２が設けられた部分と枠体２２が設けられていない部分との境界面のしなりが貫通孔Ｓによって許容される。これにより、当該境界面で基板２１が割れることが抑制される。 As shown in FIG. 3, the frame body 22 is bonded to the surface (one surface 21a) of the substrate 21 through the adhesive layer G. As a material for the adhesive layer G, for example, an adhesive material with a low emission gas such as a low melting glass or a vacuum adhesive can be used. In the present embodiment, as an example, the frame body 22 is bonded to the substrate 21 so as to overlap a portion where the through hole S is provided on the one surface 21 a of the substrate 21. For this reason, bending of the boundary surface between the portion of the substrate 21 where the frame body 22 is provided and the portion where the frame body 22 is not provided is allowed by the through hole S. Thereby, it is suppressed that the board | substrate 21 breaks in the said boundary surface.
　枠体２２は、基板２１と略同等の熱膨張係数を有する。枠体２２は、例えば基板２１と同様の組成を有するセラミック部材等である。枠体２２は、例えばガラスや金属等である。このように基板２１及び枠体２２の熱膨張係数を近づけることにより、温度変化に起因する変形（例えば熱膨張時における基板２１及び枠体２２のひずみ）等を防止することができる。 The frame body 22 has a thermal expansion coefficient substantially equal to that of the substrate 21. The frame 22 is, for example, a ceramic member having the same composition as the substrate 21. The frame 22 is, for example, glass or metal. In this manner, by making the thermal expansion coefficients of the substrate 21 and the frame body 22 close to each other, deformation (for example, distortion of the substrate 21 and the frame body 22 during thermal expansion) and the like due to a temperature change can be prevented.
　図３及び図５に示すように、試料支持体２は、基板２１の一面２１ａ、他面２１ｂ、及び貫通孔Ｓの内面、並びに枠体２２の表面を覆う導電層２３を備える。導電層２３は、絶縁性の基板２１に導電性を付与するために設けられた導電性材料からなる層である。ただし、基板２１が導電性材料からなる場合であっても導電層２３を設けることは妨げられない。導電層２３の材料としては、以下に述べる理由により、試料１０との親和性（反応性）が低く、導電性が高い金属が好ましい。 As shown in FIGS. 3 and 5, the sample support 2 includes a conductive layer 23 that covers one surface 21 a, the other surface 21 b, the inner surface of the through-hole S, and the surface of the frame body 22. The conductive layer 23 is a layer made of a conductive material provided to impart conductivity to the insulating substrate 21. However, even when the substrate 21 is made of a conductive material, the provision of the conductive layer 23 is not hindered. As a material of the conductive layer 23, a metal having low affinity (reactivity) with the sample 10 and high conductivity is preferable for the following reason.
　例えばタンパク質等の試料１０と親和性が高いＣｕ（銅）等の金属で導電層２３を形成した場合、後述する試料１０のイオン化の過程において、試料分子にＣｕ原子が付着した状態で試料１０がイオン化する場合がある。すなわち、検出器３によって検出された試料イオン１１の分子量を測定する際に、付着したＣｕの質量分だけ実際の試料１０の分子量からずれてしまうため、正確な測定が行えなくなってしまう。従って、導電層２３の材料としては、試料１０との親和性が低い金属が好ましい。 For example, when the conductive layer 23 is formed of a metal such as Cu (copper) having a high affinity with the sample 10 such as protein, the sample 10 is in a state in which Cu atoms are attached to the sample molecule in the ionization process of the sample 10 described later. May ionize. That is, when the molecular weight of the sample ions 11 detected by the detector 3 is measured, the molecular weight of the attached sample is deviated from the actual molecular weight of the sample 10, so that accurate measurement cannot be performed. Therefore, the material of the conductive layer 23 is preferably a metal having a low affinity with the sample 10.
　一方、導電性の高い金属の方が一定の電圧を容易に且つ安定して付与することができる。このため、導電性が高い金属を導電層２３とすることにより、上述したグランド電極と基板２１との間に一定の電位差を生じさせるために基板２１に一定の電圧をかけることが容易となる。また、導電性の高い金属ほど熱伝導性も高い傾向にあるので、基板２１に照射されたレーザのエネルギーを、導電層２３を介して試料１０に効率的に伝えることが可能となる。従って、導電層２３の材料としては、導電性の高い金属が好ましい。 On the other hand, a highly conductive metal can apply a constant voltage more easily and stably. For this reason, by using a highly conductive metal as the conductive layer 23, it is easy to apply a constant voltage to the substrate 21 in order to generate a constant potential difference between the ground electrode and the substrate 21 described above. Further, since the metal having higher conductivity tends to have higher thermal conductivity, the energy of the laser irradiated on the substrate 21 can be efficiently transmitted to the sample 10 through the conductive layer 23. Therefore, the material of the conductive layer 23 is preferably a highly conductive metal.
　上記観点から、導電層２３の材料としては、例えばＡｕ（金）及びＰｔ（白金）等が用いられる。例えば、導電層２３は、メッキ法、原子層堆積法（ＡＬＤ：Atomic　Layer　Deposition）、蒸着法、及びスパッタ法等を用いて、Ａｕ又はＰｔを基板２１の一面２１ａ、他面２１ｂ、及び貫通孔Ｓの内面、並びに枠体２２の表面に成膜することによって形成することができる。なお、導電層２３の材料としては、上述したＡｕ及びＰｔ以外にも、例えばＣｒ（クロム）、Ｎｉ（ニッケル）、及びＴｉ（チタン）等を用いることもできる。 From the above viewpoint, for example, Au (gold) and Pt (platinum) are used as the material of the conductive layer 23. For example, the conductive layer 23 is made of Au or Pt by using a plating method, an atomic layer deposition (ALD) method, a vapor deposition method, a sputtering method, or the like on one surface 21a, the other surface 21b, and the through hole of the substrate 21. It can be formed by forming a film on the inner surface of S and the surface of the frame body 22. In addition to Au and Pt described above, for example, Cr (chromium), Ni (nickel), Ti (titanium), or the like can be used as the material of the conductive layer 23.
　図４は、試料支持体２の実効領域Ｒの拡大平面図である。図４において、黒色の部分は貫通孔Ｓを示し、白色の部分は貫通孔Ｓが形成されていない隔壁部分を示す。図４に示すように、基板２１の表面には、略一定の大きさの貫通孔Ｓが複数形成されている。複数の貫通孔Ｓは、後述する毛細管現象による試料１０の他面２１ｂから一面２１ａへの移動（上昇）が可能な大きさに形成されていればよい。図４の例のように、貫通孔Ｓの大きさが不揃いになっていてもよいし、複数の貫通孔Ｓ同士が互いに連結している部分が存在していてもよい。実効領域Ｒにおける貫通孔Ｓの開口率（貫通孔Ｓが形成されている部分の面積／全体面積）は、実用上１０～８０％であり、特に６０～８０％であることが好ましい。 FIG. 4 is an enlarged plan view of the effective region R of the sample support 2. In FIG. 4, a black part shows the through-hole S, and a white part shows the partition part in which the through-hole S is not formed. As shown in FIG. 4, a plurality of through holes S having a substantially constant size are formed on the surface of the substrate 21. The plurality of through-holes S only need to be formed in a size that allows movement (rise) from the other surface 21b of the sample 10 to the one surface 21a by capillary action described later. As in the example of FIG. 4, the sizes of the through holes S may be uneven, or there may be portions where the plurality of through holes S are connected to each other. The aperture ratio of the through-hole S in the effective region R (the area of the portion where the through-hole S is formed / the total area) is practically 10 to 80%, particularly preferably 60 to 80%.
　図５に示すように、貫通孔Ｓは、基板２１の一面２１ａ側から他面２１ｂ側に延在している。貫通孔Ｓの幅ｄ３は、１～７００ｎｍである。また、導電層２３の厚さｄ４は、例えば１～２５ｎｍ程度である。ここで、貫通孔Ｓの幅ｄ３は、貫通孔Ｓ内に導電層２３が形成された後の孔幅である。孔幅が１～７００ｎｍの貫通孔Ｓを有する基板２１を用いることにより、上述した毛細管現象による試料１０の移動をよりスムーズに行わせることができる。本実施形態のように貫通孔Ｓの断面形状が略円形である場合、貫通孔Ｓの幅ｄ３とは、孔の径を意味する。一方、貫通孔Ｓの断面形状が円形以外の場合には、貫通孔Ｓの幅とは、貫通孔Ｓに収まる仮想的な円筒の径（有効径）を意味する。 As shown in FIG. 5, the through-hole S extends from the one surface 21a side of the substrate 21 to the other surface 21b side. The width d3 of the through hole S is 1 to 700 nm. Further, the thickness d4 of the conductive layer 23 is, for example, about 1 to 25 nm. Here, the width d3 of the through hole S is a hole width after the conductive layer 23 is formed in the through hole S. By using the substrate 21 having the through hole S with a hole width of 1 to 700 nm, the sample 10 can be moved more smoothly by the capillary phenomenon described above. When the cross-sectional shape of the through hole S is substantially circular as in the present embodiment, the width d3 of the through hole S means the diameter of the hole. On the other hand, when the cross-sectional shape of the through-hole S is other than a circle, the width of the through-hole S means a virtual cylindrical diameter (effective diameter) that fits in the through-hole S.
　次に、図３及び図６を用いて、試料支持体２の製造工程について説明する。まず、図６を用いて、基板２１の製造工程について説明する。図６の（ａ）に示すように、基板２１の材料となるＡｌ基板５０が用意される。続いて、図６の（ｂ）に示すように、Ａｌ基板５０を陽極酸化することにより、Ａｌ基板５０が表面から酸化され、複数の凹部５１ａを有する陽極酸化皮膜５１が形成される。続いて、図６の（ｃ）に示すように、陽極酸化皮膜５１をＡｌ基板５０から剥離し、剥離された陽極酸化皮膜５１の底部５１ｂを除去或いは貫通処理することにより、一面２１ａから他面２１ｂにかけて貫通する複数の貫通孔Ｓが設けられた基板２１が得られる。 Next, the manufacturing process of the sample support 2 will be described with reference to FIGS. First, the manufacturing process of the board | substrate 21 is demonstrated using FIG. As shown in FIG. 6A, an Al substrate 50 as a material for the substrate 21 is prepared. Subsequently, as shown in FIG. 6B, by anodizing the Al substrate 50, the Al substrate 50 is oxidized from the surface, and an anodized film 51 having a plurality of recesses 51a is formed. Subsequently, as shown in FIG. 6C, the anodic oxide film 51 is peeled from the Al substrate 50, and the bottom 51b of the peeled anodic oxide film 51 is removed or subjected to a penetration treatment, so that the one surface 21a is changed to the other surface. A substrate 21 provided with a plurality of through holes S penetrating through 21b is obtained.
　上述のようにして基板２１が製造された後、基板２１の外縁部に、低融点ガラスや真空用接着剤等の接着層Ｇを介して枠体２２が取り付けられる。これにより、図３に示した試料支持体２のうち導電層２３が形成される前の状態となる。最後に、基板２１の一面２１ａ、他面２１ｂ、及び貫通孔Ｓの内面、並びに枠体２２の表面を覆うように、Ａｕ又はＰｔからなる導電層２３が設けられる。上述した通り、導電層２３は、メッキ法や原子層堆積法等で、Ａｕ又はＰｔを基板２１の一面２１ａ、他面２１ｂ、及び貫通孔Ｓの内面、並びに枠体２２の表面に成膜することによって形成される。以上により、図３に示した試料支持体２が製造される。 After the substrate 21 is manufactured as described above, the frame body 22 is attached to the outer edge portion of the substrate 21 via an adhesive layer G such as low melting point glass or a vacuum adhesive. Thereby, it will be in the state before the conductive layer 23 is formed among the sample support bodies 2 shown in FIG. Finally, a conductive layer 23 made of Au or Pt is provided so as to cover one surface 21a, the other surface 21b, the inner surface of the through hole S, and the surface of the frame body 22 of the substrate 21. As described above, the conductive layer 23 is formed by depositing Au or Pt on the one surface 21a, the other surface 21b, the inner surface of the through hole S, and the surface of the frame body 22 by a plating method, an atomic layer deposition method, or the like. Formed by. Thus, the sample support 2 shown in FIG. 3 is manufactured.
　なお、上記のＡｌの陽極酸化処理においては、基板２１の厚さｄ１が１～５０μｍとなり、貫通孔Ｓの幅ｄ３が１～７００ｎｍとなるように調節される。具体的には、最初に用意するＡｌ基板５０の厚みや陽極酸化における温度及び電圧等の条件を適切に設定することにより、基板２１の厚さｄ１及び貫通孔Ｓの幅ｄ３は、予め定められた大きさ（上記範囲に含まれる大きさ）に形成される。 In the Al anodic oxidation process, the thickness d1 of the substrate 21 is adjusted to 1 to 50 μm, and the width d3 of the through hole S is adjusted to 1 to 700 nm. Specifically, the thickness d1 of the substrate 21 and the width d3 of the through hole S are determined in advance by appropriately setting the conditions such as the thickness of the Al substrate 50 prepared first and the temperature and voltage in anodization. It is formed in a size (a size included in the above range).
　次に、図７～図９を用いて、試料支持体２を用いた質量分析方法の手順について説明する。なお、図７～図９においては、導電層２３、貫通孔Ｓ、及び接着層Ｇの図示は省略している。 Next, the procedure of the mass spectrometry method using the sample support 2 will be described with reference to FIGS. 7 to 9, illustration of the conductive layer 23, the through hole S, and the adhesive layer G is omitted.
　まず、図９を用いて、試料支持体２を用いた質量分析を実行する質量分析装置１００について説明する。質量分析装置１００は、試料１０が載置される試料台１と、レーザ照射部４と、検出器（検出部）３と、を備える。 First, a mass spectrometer 100 that performs mass spectrometry using the sample support 2 will be described with reference to FIG. The mass spectrometer 100 includes a sample stage 1 on which a sample 10 is placed, a laser irradiation unit 4, and a detector (detection unit) 3.
　レーザ照射部４は、試料台１に載置された試料１０上に試料支持体２が配置された状態で、照射位置を変えながら一面２１ａにレーザＬを照射する。ここで、試料支持体２は、他面２１ｂが導電層２３を介して試料１０と接触するように、試料１０上に載置される。レーザ照射部４によって照射されるレーザＬは、例えば波長３３７ｎｍの窒素レーザ（Ｎ２レーザ）等の紫外線レーザである。 The laser irradiation unit 4 irradiates the surface 21 a with the laser L while changing the irradiation position in a state where the sample support 2 is arranged on the sample 10 placed on the sample stage 1. Here, the sample support 2 is placed on the sample 10 such that the other surface 21 b comes into contact with the sample 10 via the conductive layer 23. The laser L irradiated by the laser irradiation unit 4 is an ultraviolet laser such as a nitrogen laser (N2 laser) having a wavelength of 337 nm, for example.
　検出器３は、レーザ照射部４によるレーザＬの照射によってイオン化された試料１０（試料イオン１１）を照射位置毎に検出する。具体的には、レーザ照射部４は、試料支持体２の実効領域Ｒを予め定められた移動幅及び移動方向に従って２次元走査し、各走査位置においてレーザＬの照射を行う。検出器３は、各走査位置でのレーザＬの照射によって発生する試料イオン１１を検出する。これにより、実効領域Ｒ上の位置毎に質量分析を行うことが可能となる。このようにして得られる試料１０中の各位置における質量分析結果を総合することにより、試料分子の二次元分布図を画像化するイメージング質量分析を行うことが可能となる。以下、図７～図９を用いて、質量分析装置１００による質量分析手順について詳細に説明する。 The detector 3 detects the sample 10 (sample ion 11) ionized by the laser L irradiation by the laser irradiation unit 4 for each irradiation position. Specifically, the laser irradiation unit 4 scans the effective region R of the sample support 2 two-dimensionally according to a predetermined movement width and movement direction, and irradiates the laser L at each scanning position. The detector 3 detects sample ions 11 generated by irradiation with the laser L at each scanning position. Thereby, it becomes possible to perform mass spectrometry for every position on the effective region R. By integrating the mass analysis results at each position in the sample 10 obtained in this way, it is possible to perform imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules. Hereinafter, the mass analysis procedure by the mass spectrometer 100 will be described in detail with reference to FIGS.
　まず、上述した試料支持体２が用意される（第１工程）。試料支持体２は、質量分析装置１００を用いて質量分析を実行する者が自ら製造することにより用意されてもよいし、試料支持体２の製造者及び販売者等から取得することにより用意されてもよい。 First, the sample support 2 described above is prepared (first step). The sample support 2 may be prepared by a person who performs mass spectrometry using the mass spectrometer 100, or may be prepared by obtaining from the manufacturer and seller of the sample support 2. May be.
　続いて、図７の（ａ）に示すように、質量分析対象となる試料１０が試料台１の載置面１ａに載置され、且つ、図７の（ｂ）に示すように、他面２１ｂが導電層２３（図３参照）を介して試料１０と接触するように試料支持体２が試料１０上に配置される（第２工程）。ここで、分析対象となる試料１０を毛細管現象によって基板２１の一面２１ａ側に移動させるために、試料支持体２は、平面視において実効領域Ｒ内に試料１０が含まれるように、試料１０上に配置される。なお、後述する毛細管現象による試料１０の移動をスムーズにするために、試料１０の粘性を低くするための溶液（例えばアセトニトリル混合液等）を試料１０に混ぜてもよい。 Subsequently, as shown in FIG. 7 (a), the sample 10 to be subjected to mass spectrometry is placed on the placement surface 1a of the sample stage 1, and as shown in FIG. The sample support 2 is disposed on the sample 10 so that 21b comes into contact with the sample 10 through the conductive layer 23 (see FIG. 3) (second step). Here, in order to move the sample 10 to be analyzed to the one surface 21a side of the substrate 21 by capillary action, the sample support 2 is placed on the sample 10 so that the sample 10 is included in the effective region R in plan view. Placed in. It should be noted that a solution for lowering the viscosity of the sample 10 (for example, an acetonitrile mixed solution) may be mixed with the sample 10 in order to make the movement of the sample 10 due to capillary action, which will be described later, smooth.
　続いて、図８の（ａ）に示すように、試料支持体２は、試料台１に固定される（第２工程続き）。ここでは一例として、カーボンテープ等の導電性を有する粘着テープＴによって、試料支持体２の四辺（枠体２２の上面及び側面、並びに基板２１の側面）が、試料台１の載置面１ａに対して固定される。このように、試料台１に対して試料支持体２を固定することにより、試料１０と試料支持体２とを密着させ、後述する毛細管現象による試料１０の移動をよりスムーズに行わせることができる。また、試料１０上に配置された試料支持体２の横滑りが防止され、試料支持体２の横滑りによって試料１０の位置情報が失われることを抑制できる。 Subsequently, as shown in FIG. 8A, the sample support 2 is fixed to the sample stage 1 (continuation of the second step). Here, as an example, the four sides of the sample support 2 (the upper surface and the side surfaces of the frame body 22 and the side surfaces of the substrate 21) are placed on the mounting surface 1a of the sample table 1 by an adhesive tape T having conductivity such as a carbon tape. It is fixed against. In this way, by fixing the sample support 2 to the sample stage 1, the sample 10 and the sample support 2 can be brought into close contact with each other, and the sample 10 can be moved more smoothly by capillary action described later. . In addition, the skidding of the sample support 2 arranged on the sample 10 is prevented, and it is possible to prevent the position information of the sample 10 from being lost due to the skidding of the sample support 2.
　ここで、試料台１が導電性を有している場合、試料台１と試料支持体２とは、導電性を有する粘着テープＴによって電気的に接続される。従って、図８の（ａ）に示すように粘着テープＴで試料支持体２を試料台１に固定した状態で、試料台１に所定の電流を流すことにより、基板２１に所定の電圧をかけることができる。これにより、上述したグランド電極と基板２１との間に一定の電位差を生じさせることができる。また、本実施形態では、導電層２３が枠体２２を覆っており、粘着テープＴが枠体２２上の導電層２３に接触しているため、試料支持体２と電源（試料台１に電流を流す所定の電源）とのコンタクトを、枠体２２上で取ることができる。すなわち、基板２１上の実効領域Ｒを減らすことなく、試料支持体２と電源とのコンタクトを取ることができる。 Here, when the sample stage 1 has conductivity, the sample stage 1 and the sample support 2 are electrically connected by the adhesive tape T having conductivity. Therefore, a predetermined voltage is applied to the substrate 21 by applying a predetermined current to the sample stage 1 in a state where the sample support 2 is fixed to the sample stage 1 with the adhesive tape T as shown in FIG. be able to. Thereby, a constant potential difference can be generated between the ground electrode and the substrate 21 described above. In this embodiment, since the conductive layer 23 covers the frame body 22 and the adhesive tape T is in contact with the conductive layer 23 on the frame body 22, the sample support 2 and the power source (the current on the sample stage 1). Contact with a predetermined power source) can be taken on the frame 22. That is, it is possible to make contact between the sample support 2 and the power source without reducing the effective area R on the substrate 21.
　図８の（ｂ）に示すように、上述のように試料支持体２が試料１０上に配置されることにより、毛細管現象によって、試料１０が基板２１の他面２１ｂ側から貫通孔Ｓを介して一面２１ａ側に向かって移動（上昇）する。そして、試料１０は、試料支持体２の一面２１ａ側に表面張力によって留まる状態となる。ここで、試料台１の載置面１ａと基板２１の一面２１ａ及び他面２１ｂとは、互いに略平行に配置される。従って、試料台１に載置された試料１０は、毛細管現象によって、試料台１の載置面１ａに直交する方向に沿って、基板２１の他面２１ｂ側から貫通孔Ｓを介して一面２１ａ側に移動することになる。これにより、毛細管現象による移動の前後において、試料１０（試料１０を構成する各試料分子）の位置情報が維持される。言い換えると、試料１０を構成する各試料分子の２次元座標（試料台１の載置面１ａと平行な２次元平面における位置）は、毛細管現象による移動前後において大きく変化しない。従って、このような毛細管現象によって、試料１０の位置情報を維持したまま、試料１０を基板２１の他面２１ｂ側から一面２１ａ側に移動させることができる。 As shown in FIG. 8B, when the sample support 2 is arranged on the sample 10 as described above, the sample 10 passes through the through-hole S from the other surface 21b side of the substrate 21 by capillary action. And move (rise) toward the surface 21a. And the sample 10 will be in the state which stays on the one surface 21a side of the sample support body 2 by surface tension. Here, the mounting surface 1a of the sample stage 1 and the one surface 21a and the other surface 21b of the substrate 21 are arranged substantially parallel to each other. Therefore, the sample 10 placed on the sample table 1 is formed on the one surface 21a through the through hole S from the other surface 21b side of the substrate 21 along the direction orthogonal to the placement surface 1a of the sample table 1 by capillary action. Will move to the side. Thereby, the position information of the sample 10 (each sample molecule constituting the sample 10) is maintained before and after the movement due to the capillary phenomenon. In other words, the two-dimensional coordinates (positions in a two-dimensional plane parallel to the mounting surface 1a of the sample table 1) of each sample molecule constituting the sample 10 do not change greatly before and after the movement due to the capillary phenomenon. Therefore, the capillary 10 can move the sample 10 from the other surface 21b side of the substrate 21 to the one surface 21a side while maintaining the position information of the sample 10.
　続いて、図９に示すように、レーザ照射部４によって、基板２１の一面２１ａにレーザＬが照射され、毛細管現象によって他面２１ｂ側から貫通孔Ｓを介して一面２１ａ側に移動した試料１０がイオン化される（第３工程）。そして、検出器３によって、イオン化された試料１０（試料イオン１１）が検出される（第４工程）。第３工程でのレーザＬの照射、及び第４工程での試料イオン１１の検出は、レーザＬの照射位置を変えながら当該照射位置毎に行われる。具体的には、レーザ照射部４が、実効領域Ｒを予め定められた移動幅及び移動方向に従って走査し、レーザＬの照射位置を変えながら、各照射位置においてレーザＬを照射する。そして、各照射位置でのレーザ照射部４によるレーザＬの照射によって真空中に放出された試料イオン１１を検出器３が検出する。その結果、各照射位置において検出された試料イオン１１の測定結果に基づいて、試料分子の二次元分布図を画像化するイメージング質量分析を行うことが可能となる。 Subsequently, as shown in FIG. 9, the laser irradiation unit 4 irradiates the surface 21a of the substrate 21 with the laser L, and the sample 10 moved from the other surface 21b side to the one surface 21a side through the through hole S due to capillary action. Is ionized (third step). Then, the ionized sample 10 (sample ion 11) is detected by the detector 3 (fourth step). The irradiation of the laser L in the third step and the detection of the sample ions 11 in the fourth step are performed for each irradiation position while changing the irradiation position of the laser L. Specifically, the laser irradiation unit 4 scans the effective region R according to a predetermined movement width and movement direction, and irradiates the laser L at each irradiation position while changing the irradiation position of the laser L. And the detector 3 detects the sample ion 11 discharge | released in the vacuum by irradiation of the laser L by the laser irradiation part 4 in each irradiation position. As a result, based on the measurement result of the sample ions 11 detected at each irradiation position, it is possible to perform imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules.
　上記表面支援レーザ脱離イオン化法（第１～第３工程）によれば、複数の貫通孔Ｓが設けられた基板２１が試料１０上に配置されることで、毛細管現象によって、基板２１の他面２１ｂ側から貫通孔Ｓを介して一面２１ａ側に向けて試料１０を上昇させることができる。これにより、試料１０の位置情報（試料１０を構成する分子の二次元分布）を維持したまま、試料１０を基板２１の他面２１ｂ側から一面２１ａ側に移動させることができる。そして、レーザＬが基板２１の一面２１ａに照射され、一面２１ａ側に移動した試料１０に導電層２３を介してエネルギーが伝達されることにより、試料１０がイオン化される。その結果、試料１０の位置情報を維持したまま試料１０のイオン化を行うことができる。従って、上記方法によれば、複数の貫通孔Ｓが設けられた基板２１を試料１０上に載置する簡単な操作によって、試料１０の位置情報を維持したまま試料１０のイオン化を行うことができる。 According to the surface-assisted laser desorption / ionization method (first to third steps), the substrate 21 provided with a plurality of through-holes S is disposed on the sample 10, so that the other of the substrate 21 is caused by capillary action. The sample 10 can be raised from the surface 21b side through the through hole S toward the one surface 21a side. Thereby, the sample 10 can be moved from the other surface 21b side of the substrate 21 to the one surface 21a side while maintaining the position information of the sample 10 (two-dimensional distribution of molecules constituting the sample 10). Then, the laser L is irradiated on the one surface 21 a of the substrate 21, and energy is transmitted to the sample 10 moved to the one surface 21 a side through the conductive layer 23, whereby the sample 10 is ionized. As a result, the sample 10 can be ionized while maintaining the position information of the sample 10. Therefore, according to the above method, the sample 10 can be ionized while maintaining the position information of the sample 10 by a simple operation of placing the substrate 21 provided with the plurality of through holes S on the sample 10. .
　また、Ａｌを陽極酸化することで複数の貫通孔Ｓが設けられた基板２１を備える試料支持体２を用いることにより、上述した毛細管現象による試料１０の移動を適切に実現することができる。ここで、Ａｌの代わりにＡｌ以外のバルブ金属又はＳｉを陽極酸化することで得られた基板２１を備える試料支持体２を用いても、同様の効果が得られる。 Further, by using the sample support 2 including the substrate 21 provided with a plurality of through holes S by anodizing Al, the movement of the sample 10 by the capillary phenomenon described above can be appropriately realized. Here, the same effect can be obtained by using the sample support 2 provided with the substrate 21 obtained by anodizing a valve metal other than Al or Si instead of Al.
　また、孔幅ｄ３が１～７００ｎｍの貫通孔Ｓを有する基板２１を用いることにより、上述した毛細管現象による試料１０の移動をよりスムーズに行わせることができる。 Further, by using the substrate 21 having the through-hole S having a hole width d3 of 1 to 700 nm, the sample 10 can be moved more smoothly by the capillary phenomenon described above.
　また、試料支持体２が、基板２１の一面２１ａの外縁部に取り付けられた枠体２２を備えているので、枠体２２によって基板２１のしなりが抑制されるとともに、試料支持体２を支持したり移動させたりする際の取り扱いが容易となる。このため、第２工程における試料支持体２の試料１０上への配置を容易に行うことができる。 Further, since the sample support 2 includes the frame 22 attached to the outer edge portion of the one surface 21a of the substrate 21, the frame 22 suppresses the bending of the substrate 21 and supports the sample support 2. Handling when moving or moving. For this reason, arrangement | positioning on the sample 10 of the sample support body 2 in a 2nd process can be performed easily.
　また、上記質量分析方法（第１～第４工程）によれば、試料１０上に試料支持体２を配置する簡単な操作で、試料１０の位置情報を維持したまま試料１０のイオン化を行うことができる。そして、レーザＬの照射位置を変えながら、イオン化された試料１０（試料イオン１１）を当該照射位置毎に検出することにより、試料分子の二次元分布を把握することができる。従って、上記質量分析方法によれば、簡単な操作で、試料分子の二次元分布図を画像化するイメージング質量分析を行うことができる。 In addition, according to the mass spectrometry method (first to fourth steps), the sample 10 is ionized while maintaining the position information of the sample 10 by a simple operation of placing the sample support 2 on the sample 10. Can do. Then, by detecting the ionized sample 10 (sample ions 11) for each irradiation position while changing the irradiation position of the laser L, the two-dimensional distribution of the sample molecules can be grasped. Therefore, according to the mass spectrometry method, imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
　また、上記質量分析装置１００によれば、試料１０上に試料支持体２を配置する簡単な操作で、試料１０の位置情報を維持したまま試料１０のイオン化を行うことができる。そして、レーザ照射部４が照射位置を変えながらレーザＬを照射し、検出器３がイオン化された試料１０（試料イオン１１）を当該照射位置毎に検出することにより、試料分子の二次元分布を把握することができる。従って、上記質量分析装置１００によれば、簡単な操作で、試料分子の二次元分布図を画像化するイメージング質量分析を行うことができる。 Further, according to the mass spectrometer 100, the sample 10 can be ionized while maintaining the position information of the sample 10 by a simple operation of placing the sample support 2 on the sample 10. Then, the laser irradiation unit 4 irradiates the laser L while changing the irradiation position, and the detector 3 detects the ionized sample 10 (sample ion 11) for each irradiation position, thereby obtaining a two-dimensional distribution of the sample molecules. I can grasp it. Therefore, according to the mass spectrometer 100, imaging mass spectrometry for imaging a two-dimensional distribution map of sample molecules can be performed with a simple operation.
　以上、本発明の実施形態について説明したが、本発明は上記実施形態に限定されるものではなく、本発明は、その要旨を逸脱しない範囲で様々な変形が可能である。 As mentioned above, although embodiment of this invention was described, this invention is not limited to the said embodiment, A various deformation | transformation is possible for this invention in the range which does not deviate from the summary.
　例えば、基板２１は、半導体等の導電性材料からなっていてもよい。この場合、試料支持体２は、基板２１に導電性を付与するための導電層２３を省略することができる。試料支持体２が導電層２３を備えない場合には、上記第２工程において、他面２１ｂが直接試料１０と接触するように試料支持体２が試料１０上に配置されることになる。このように基板２１が導電性材料からなり、導電層２３が省略された試料支持体２を用いる場合にも、上述した導電層２３を備える試料支持体２を用いる場合と同様の効果を得ることができる。 For example, the substrate 21 may be made of a conductive material such as a semiconductor. In this case, the sample support 2 can omit the conductive layer 23 for imparting conductivity to the substrate 21. When the sample support 2 does not include the conductive layer 23, the sample support 2 is arranged on the sample 10 so that the other surface 21b is in direct contact with the sample 10 in the second step. As described above, even when the sample support 2 in which the substrate 21 is made of a conductive material and the conductive layer 23 is omitted is used, the same effect as in the case of using the sample support 2 including the conductive layer 23 described above can be obtained. Can do.
　また、上記表面支援レーザ脱離イオン化法（第１～第３工程）による試料１０のイオン化は、本実施形態で説明した試料１０のイメージング質量分析のみならず、イオンモビリティ測定等の他の測定・実験にも利用できる。 In addition, the ionization of the sample 10 by the surface-assisted laser desorption ionization method (first to third steps) is not limited to the imaging mass spectrometry of the sample 10 described in the present embodiment, but other measurements such as ion mobility measurement. It can also be used for experiments.
　また、導電層２３は、少なくとも基板２１の一面２１ａを覆うように、蒸着等によって設けられてもよい。すなわち、導電層２３は、基板２１の他面２１ｂ及び貫通孔Ｓの内面に設けられなくともよい。この場合、上記第２工程において、他面２１ｂが試料１０に対向するように試料支持体２が試料１０上に配置され、他面２１ｂが直接試料１０に接触する。また、導電層２３が少なくとも基板２１の一面２１ａ及び枠体２２の表面を覆うように設けられていれば、基板２１と電極とのコンタクトを枠体２２上で取ることができる。 Further, the conductive layer 23 may be provided by vapor deposition or the like so as to cover at least the one surface 21a of the substrate 21. That is, the conductive layer 23 may not be provided on the other surface 21b of the substrate 21 and the inner surface of the through hole S. In this case, in the second step, the sample support 2 is disposed on the sample 10 so that the other surface 21b faces the sample 10, and the other surface 21b directly contacts the sample 10. Further, if the conductive layer 23 is provided so as to cover at least one surface 21 a of the substrate 21 and the surface of the frame body 22, contact between the substrate 21 and the electrode can be made on the frame body 22.
　図１０～図１３は、貫通孔Ｓの孔幅と上記質量分析方法によって測定されたマススペクトルとの関係を示す。ここで、試料支持体としては、導電層２３（ここではＰｔ）を基板２１の他面２１ｂ及び貫通孔Ｓの内面には設けずに一面２１ａ及び枠体２２の表面を覆うように設けたものを用いた。また、基板２１の厚さｄ１は１０μｍであり、測定対象の試料は「質量電荷比（ｍ／ｚ）＝１０４９」のペプチドである。図１０～図１３において、（ａ）は貫通孔Ｓの孔幅を５０ｎｍとした場合の測定結果であり、（ｂ）は貫通孔Ｓの孔幅を１００ｎｍとした場合の測定結果であり、（ｃ）は貫通孔Ｓの孔幅を２００ｎｍとした場合の測定結果であり、（ｄ）は貫通孔Ｓの孔幅を３００ｎｍとした場合の測定結果であり、（ｅ）は貫通孔Ｓの孔幅を４００ｎｍとした場合の測定結果であり、（ｆ）は貫通孔Ｓの孔幅を５００ｎｍとした場合の測定結果であり、（ｇ）は貫通孔Ｓの孔幅を６００ｎｍとした場合の測定結果であり、（ｈ）は貫通孔Ｓの孔幅を７００ｎｍとした場合の測定結果である。図１０～図１３において、縦軸は、ピーク値を１００（％）として規格化された信号強度（Intensity）を示す。 10 to 13 show the relationship between the hole width of the through hole S and the mass spectrum measured by the mass spectrometry method. Here, as the sample support, the conductive layer 23 (here, Pt) is not provided on the other surface 21b of the substrate 21 and the inner surface of the through-hole S, but is provided so as to cover the surface 21a and the surface of the frame body 22. Was used. The thickness d1 of the substrate 21 is 10 μm, and the sample to be measured is a peptide with “mass-to-charge ratio (m / z) = 1049”. 10 to 13, (a) is a measurement result when the hole width of the through hole S is 50 nm, and (b) is a measurement result when the hole width of the through hole S is 100 nm. c) is a measurement result when the hole width of the through-hole S is 200 nm, (d) is a measurement result when the hole width of the through-hole S is 300 nm, and (e) is a hole of the through-hole S. (F) is a measurement result when the hole width of the through-hole S is 500 nm, and (g) is a measurement result when the hole width of the through-hole S is 600 nm. It is a result and (h) is a measurement result when the hole width of the through-hole S is 700 nm. 10 to 13, the vertical axis represents the signal intensity (Intensity) normalized with the peak value set to 100 (%).
　図１０～図１３に示すように、基板２１の貫通孔Ｓの孔幅が５０ｎｍ、１００ｎｍ、２００ｎｍ、３００ｎｍ、４００ｎｍ、５００ｎｍ、６００ｎｍ、及び７００ｎｍのいずれの場合にも、ピークを観測可能な適切なスペクトルが得られた。このように、少なくとも一面２１ａに導電層２３が設けられた基板２１を備える試料支持体を用いることで、質量分析を適切に行うことができる。 As shown in FIG. 10 to FIG. 13, it is possible to observe a peak when the through-hole S of the substrate 21 has a hole width of 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, and 700 nm. A spectrum was obtained. Thus, mass spectrometry can be appropriately performed by using the sample support including the substrate 21 provided with the conductive layer 23 on at least one surface 21a.
　図１４は、基板２１の厚さｄ１（Thickness）と上記質量分析方法によって測定されたピークの信号強度との関係を示す。図１４において、縦軸は、基板２１の厚さｄ１が１０μｍのときの信号強度を“１”とした場合の相対的な信号強度（Relative　intensity）である。ここで、試料支持体としては、上述のように導電層２３（ここではＰｔ）を基板２１の他面２１ｂ及び貫通孔Ｓの内面には設けずに一面２１ａ及び枠体２２の表面を覆うように設けたものを用いた。貫通孔Ｓの孔幅は２００ｎｍである。また、測定対象の試料は「質量電荷比（ｍ／ｚ）＝１０４９」のペプチドである。 FIG. 14 shows the relationship between the thickness d1 (Thickness) of the substrate 21 and the signal intensity of the peak measured by the mass spectrometry method. In FIG. 14, the vertical axis represents the relative signal intensity when the signal intensity when the thickness d1 of the substrate 21 is 10 μm is “1”. Here, as the sample support, the conductive layer 23 (here, Pt) is not provided on the other surface 21b of the substrate 21 and the inner surface of the through-hole S as described above so as to cover the one surface 21a and the surface of the frame 22. What was provided in was used. The hole width of the through hole S is 200 nm. The sample to be measured is a peptide with “mass-to-charge ratio (m / z) = 1049”.
　上記測定結果において、基板２１の厚さｄ１が１０μｍの場合における信号強度は、質量分析において十分な大きさであった。また、図１４に示すように、基板２１の厚さｄ１が小さい程信号強度が大きくなる傾向があり、基板２１の厚さｄ１が３～１０μｍの範囲にある場合に、十分な信号強度が得られた。一方、基板強度を確保する観点からは、基板２１の厚さｄ１は大きい方がよい。このことから、基板２１の厚さｄ１は、５～１０μｍとされてもよい。これにより、基板２１の強度を保つとともに、質量分析において十分な信号強度を得ることができる。 In the above measurement results, the signal intensity when the thickness d1 of the substrate 21 was 10 μm was sufficiently large in mass spectrometry. Further, as shown in FIG. 14, the signal strength tends to increase as the thickness d1 of the substrate 21 decreases, and sufficient signal strength is obtained when the thickness d1 of the substrate 21 is in the range of 3 to 10 μm. It was. On the other hand, from the viewpoint of ensuring the substrate strength, the thickness d1 of the substrate 21 is preferably large. Therefore, the thickness d1 of the substrate 21 may be 5 to 10 μm. Thereby, while maintaining the intensity | strength of the board | substrate 21, sufficient signal intensity | strength can be obtained in mass spectrometry.
　また、上記実施形態では、試料支持体２の枠体２２を粘着テープＴによって試料台１に固定する形態について説明したが、試料支持体２を試料台１に固定する形態は上記形態に限定されない。以下、図１５～図１７を用いて、試料支持体２の第１～第３の変形例とともに、試料支持体２を試料台１に固定する形態のバリエーションについて説明する。なお、図１５～図１７においては、導電層２３及び貫通孔Ｓの図示を省略している。また、図１６及び図１７においては、枠体と基板とを接着する接着層Ｇの図示についても省略している。 Moreover, although the said embodiment demonstrated the form which fixes the frame 22 of the sample support body 2 to the sample stand 1 with the adhesive tape T, the form which fixes the sample support body 2 to the sample stand 1 is not limited to the said form. . Hereinafter, variations of the embodiment in which the sample support 2 is fixed to the sample stage 1 will be described together with the first to third modifications of the sample support 2 with reference to FIGS. 15 to 17, illustration of the conductive layer 23 and the through hole S is omitted. 16 and 17, the illustration of the adhesive layer G that bonds the frame and the substrate is also omitted.
（第１の変形例）
　図１５に示すように、第１の変形例に係る試料支持体２Ａは、基板２１に枠体２２が設けられておらず、基板２１の一面２１ａに粘着テープＴが直接貼り付けられている点で、試料支持体２と主に相違する。粘着テープＴは、粘着面Ｔａが基板２１の一面２１ａに対向し、且つ、基板２１の外縁よりも外側に延在する部分を有するように、一面２１ａの外縁部に貼り付けられている。これにより、図１５に示すように、粘着面Ｔａを基板２１の外縁及び試料台１の載置面１ａに貼り付けることができる。その結果、試料支持体２Ａは、粘着テープＴによって試料台１に対して固定される。試料支持体２Ａによれば、例えば表面に凹凸を有する試料１０の質量分析を行う場合等において、試料１０に対する基板２１の追従性を向上させることができる。 (First modification)
As shown in FIG. 15, the sample support 2 </ b> A according to the first modification has a frame 22 not provided on the substrate 21, and the adhesive tape T is directly attached to one surface 21 a of the substrate 21. Thus, it is mainly different from the sample support 2. The adhesive tape T is affixed to the outer edge portion of the one surface 21 a so that the adhesive surface Ta faces the one surface 21 a of the substrate 21 and has a portion extending outward from the outer edge of the substrate 21. As a result, as shown in FIG. 15, the adhesive surface Ta can be attached to the outer edge of the substrate 21 and the mounting surface 1 a of the sample stage 1. As a result, the sample support 2 </ b> A is fixed to the sample table 1 by the adhesive tape T. According to the sample support 2A, the followability of the substrate 21 with respect to the sample 10 can be improved, for example, when performing mass analysis of the sample 10 having an uneven surface.
　また、試料台１が導電性を有している場合、試料台１と試料支持体２Ａ（具体的には、基板２１の一面２１ａ上に設けられた導電層２３）とは、導電性を有する粘着テープＴを介して電気的に接続される。従って、図１５に示すように粘着テープＴを介して試料支持体２を試料台１に固定した状態で、試料台１に所定の電流を流すことにより、基板２１に所定の電圧をかけることができる。 When the sample stage 1 has conductivity, the sample stage 1 and the sample support 2A (specifically, the conductive layer 23 provided on the one surface 21a of the substrate 21) have conductivity. It is electrically connected via an adhesive tape T. Accordingly, as shown in FIG. 15, a predetermined voltage is applied to the substrate 21 by applying a predetermined current to the sample stage 1 with the sample support 2 fixed to the sample stage 1 via the adhesive tape T. it can.
　なお、試料支持体２Ａは、基板２１の外縁に粘着テープＴが貼り付けられ、且つ、基板２１の外縁よりも外側に延在する部分の粘着面Ｔａに粘着保護シートが設けられた状態で流通させられてもよい。この場合、試料支持体２Ａの使用者は、試料支持体２Ａを試料台１に固定する直前に粘着保護シートを剥がし、粘着面Ｔａを載置面１ａに貼り付けることで、試料１０の質量分析の準備を容易に行うことができる。 The sample support 2 </ b> A is distributed in a state where the adhesive tape T is attached to the outer edge of the substrate 21, and the adhesive protective sheet is provided on the adhesive surface Ta of the portion extending outside the outer edge of the substrate 21. May be allowed. In this case, the user of the sample support 2A peels off the adhesive protective sheet immediately before fixing the sample support 2A to the sample stage 1, and attaches the adhesive surface Ta to the mounting surface 1a, whereby mass analysis of the sample 10 is performed. Can be easily prepared.
（第２の変形例）
　図１６に示すように、第２の変形例に係る試料支持体２Ｂは、基板２１の外縁よりも外側に延在する部分を有する枠体１２２を備える点で、試料支持体２と主に相違する。このような枠体１２２により、試料支持体２Ｂを持ち運ぶ際等において、基板２１の端部の破損を適切に抑制することができる。さらに、図１６に示すように、枠体１２２において基板２１の外縁よりも外側に延在する部分には、ネジ３０を挿通させるための挿通孔１２２ａが設けられている。この場合、例えば挿通孔１２２ａに対応する位置にネジ孔１ｂを有する試料台１Ａを用いることで、ネジ留めによって試料支持体２Ｂを試料台１Ａに確実に固定することができる。具体的には、挿通孔１２２ａ及びネジ孔１ｂにネジ３０を挿通させることで、試料支持体２Ｂを試料台１Ａに固定することができる。 (Second modification)
As shown in FIG. 16, the sample support 2 </ b> B according to the second modification is mainly different from the sample support 2 in that it includes a frame body 122 having a portion extending outside the outer edge of the substrate 21. To do. Such a frame body 122 can appropriately suppress breakage of the end portion of the substrate 21 when carrying the sample support 2B. Further, as shown in FIG. 16, an insertion hole 122 a through which the screw 30 is inserted is provided in a portion of the frame body 122 that extends outside the outer edge of the substrate 21. In this case, for example, by using the sample stage 1A having the screw hole 1b at a position corresponding to the insertion hole 122a, the sample support 2B can be securely fixed to the sample stage 1A by screwing. Specifically, the sample support 2B can be fixed to the sample stage 1A by inserting the screw 30 through the insertion hole 122a and the screw hole 1b.
　また、試料台１Ａが導電性を有し、且つ、ネジ３０が導電性を有する場合、試料台１Ａと試料支持体２Ｂ（具体的には、枠体１２２の表面に形成された導電層２３）とは、ネジ３０を介して電気的に接続される。従って、図１６に示すようにネジ３０を介して試料支持体２Ｂを試料台１Ａに固定した状態で、試料台１Ａに所定の電流を流すことにより、基板２１に所定の電圧をかけることができる。 In addition, when the sample stage 1A has conductivity and the screw 30 has conductivity, the sample stage 1A and the sample support 2B (specifically, the conductive layer 23 formed on the surface of the frame body 122). Is electrically connected via a screw 30. Accordingly, a predetermined voltage can be applied to the substrate 21 by flowing a predetermined current through the sample stage 1A in a state where the sample support 2B is fixed to the sample stage 1A via the screw 30 as shown in FIG. .
（第３の変形例）
　図１７に示すように、第３の変形例に係る試料支持体２Ｃは、基板２１の他面２１ｂの外縁部に設けられ、一面２１ａから他面２１ｂに向かう方向を向く粘着面２４ａを有する粘着層２４を備える点で、試料支持体２と主に相違する。粘着層２４は、例えば測定対象の試料１０の厚みに応じて予め設定された厚さを有する両面テープ等である。例えば、粘着層２４の一方の粘着面２４ｂは、予め基板２１の他面２１ｂの外縁部に貼り付けられており、粘着層２４の他方の粘着面２４ａは、試料支持体２Ｃを試料台１に固定する際に、載置面１ａに貼り付けられる。試料支持体２Ｃによれば、試料支持体２Ｃを試料台１に固定する構成を単純化することができる。 (Third Modification)
As shown in FIG. 17, the sample support 2C according to the third modification is provided on the outer edge portion of the other surface 21b of the substrate 21, and has an adhesive surface 24a facing in the direction from the one surface 21a to the other surface 21b. The main difference from the sample support 2 is that the layer 24 is provided. The adhesive layer 24 is, for example, a double-sided tape having a thickness set in advance according to the thickness of the sample 10 to be measured. For example, one adhesive surface 24 b of the adhesive layer 24 is previously attached to the outer edge portion of the other surface 21 b of the substrate 21, and the other adhesive surface 24 a of the adhesive layer 24 attaches the sample support 2 C to the sample stage 1. When fixing, it is affixed on the mounting surface 1a. According to the sample support 2C, the configuration for fixing the sample support 2C to the sample stage 1 can be simplified.
　また、試料台１が導電性を有し、且つ、粘着層２４が導電性を有する場合、試料台１と試料支持体２Ｃ（具体的には、基板２１）とは、粘着層２４を介して電気的に接続される。従って、図１７に示すように粘着層２４を介して試料支持体２Ｃを試料台１に固定した状態で、試料台１に所定の電流を流すことにより、基板２１に所定の電圧をかけることができる。 When the sample stage 1 has conductivity and the adhesive layer 24 has conductivity, the sample stage 1 and the sample support 2C (specifically, the substrate 21) are interposed via the adhesive layer 24. Electrically connected. Accordingly, as shown in FIG. 17, a predetermined voltage is applied to the substrate 21 by flowing a predetermined current through the sample stage 1 with the sample support 2 </ b> C fixed to the sample stage 1 via the adhesive layer 24. it can.
　なお、試料支持体２Ｃは、粘着層２４の粘着面２４ｂが基板２１の他面２１ｂの外縁部に貼り付けられ、且つ、粘着面２４ａに粘着保護シートが設けられた状態で流通させられてもよい。この場合、試料支持体２Ｃの使用者は、試料支持体２Ｃを試料台１に固定する直前に粘着保護シートを剥がし、粘着面２４ａを載置面１ａに貼り付けることで、試料１０の質量分析の準備を容易に行うことができる。 The sample support 2C may be circulated with the adhesive surface 24b of the adhesive layer 24 attached to the outer edge portion of the other surface 21b of the substrate 21 and the adhesive protective sheet provided on the adhesive surface 24a. Good. In this case, the user of the sample support 2C peels off the adhesive protective sheet immediately before fixing the sample support 2C to the sample stage 1, and attaches the adhesive surface 24a to the mounting surface 1a, whereby mass analysis of the sample 10 is performed. Can be easily prepared.
　また、上記実施形態及び変形例に係る試料支持体２，２Ａ，２Ｂ，２Ｃは、導電層２３が形成された後に、焼成されてもよい。すなわち、上記実施形態における試料支持体の製造工程において、導電層２３が形成された後の試料支持体を焼成する焼成工程が含まれてもよい。枠体２２が設けられる場合には、基板２１、枠体２２、及び導電層２３を備える試料支持体に対して焼成工程が実施される。また、枠体２２が省略される場合には、基板２１及び導電層２３を備える試料支持体に対して焼成工程が実施される。 Further, the sample supports 2, 2A, 2B, and 2C according to the above-described embodiments and modifications may be baked after the conductive layer 23 is formed. That is, the manufacturing process of the sample support in the above embodiment may include a firing step of firing the sample support after the conductive layer 23 is formed. When the frame body 22 is provided, a baking process is performed on the sample support including the substrate 21, the frame body 22, and the conductive layer 23. When the frame body 22 is omitted, a baking process is performed on the sample support including the substrate 21 and the conductive layer 23.
　このような焼成工程の実施により、導電層２３（例えばＰｔ）の結晶性を向上させることができ、質量分析に一層適した試料支持体を得ることができる。ここで、試料支持体の焼成は、焼成後の導電層２３（試料支持体）に対するＸ線回折（ＸＲＤ：X-ray　diffraction）測定において、当該導電層２３を形成する導電性材料（ここではＰｔ）の結晶の回折ピークが示されるように、実施されるのが好ましい。ここで、「導電性材料の結晶の回折ピークを示す」とは、焼成前の試料支持体に対するＸ線回折測定によって得られる測定結果よりも、導電性材料の結晶の回折パターン（ピーク強度等）を明確に示すことを意味する。 By carrying out such a baking step, the crystallinity of the conductive layer 23 (for example, Pt) can be improved, and a sample support more suitable for mass spectrometry can be obtained. Here, the firing of the sample support is performed by conducting a conductive material (here, Pt) in the X-ray diffraction (XRD) measurement on the conductive layer 23 (sample support) after firing. It is preferably carried out so that a diffraction peak of the crystal of Here, “shows the diffraction peak of the crystal of the conductive material” means that the diffraction pattern (peak intensity, etc.) of the crystal of the conductive material is higher than the measurement result obtained by the X-ray diffraction measurement on the sample support before firing. Is clearly indicated.
　図１８の（ａ）は、焼成前の試料支持体を有する質量分析装置１００によって測定されたマススペクトルを示す。一方、図１８の（ｂ）は、焼成温度４００℃での焼成後の試料支持体を有する質量分析装置１００によって測定されたマススペクトルを示す。なお、図１８の（ａ）及び（ｂ）の間で、焼成の有無以外の測定条件（試料の種類及び試料支持体の構成等）は同一である。また、図１８の（ａ）及び（ｂ）の縦軸は、焼成後の試料支持体を用いた場合のピークの信号強度（すなわち、図１８の（ｂ）のグラフのピーク値）を“１００”とした場合の相対的な信号強度を示す。図１８に示すように、焼成後の試料支持体を用いることにより、焼成前の試料支持体を用いる場合よりも、質量分析における信号強度を向上させることができる。このように、上述の焼成工程を実施することにより、質量分析に一層適した試料支持体を得ることができる。 (A) of FIG. 18 shows a mass spectrum measured by the mass spectrometer 100 having the sample support before firing. On the other hand, FIG. 18B shows a mass spectrum measured by a mass spectrometer 100 having a sample support after firing at a firing temperature of 400 ° C. 18A and 18B, the measurement conditions other than the presence or absence of firing (the type of sample and the configuration of the sample support, etc.) are the same. Also, the vertical axes of FIGS. 18A and 18B indicate the peak signal intensity (that is, the peak value in the graph of FIG. 18B) when the sample support after firing is used as “100”. "Indicates the relative signal strength. As shown in FIG. 18, by using the sample support after firing, the signal intensity in mass spectrometry can be improved as compared with the case of using the sample support before firing. In this way, a sample support more suitable for mass spectrometry can be obtained by performing the above-described firing step.
　１…試料台、２，２Ａ，２Ｂ，２Ｃ…試料支持体、３…検出器、４…レーザ照射部、１０…試料、１１…試料イオン、２１…基板、２１ａ…一面、２１ｂ…他面、２２，１２２…枠体、２３…導電層、２４…粘着層、２４ａ，２４ｂ…粘着面、３０…ネジ、１２２ａ…挿通孔、Ｌ…レーザ、Ｓ…貫通孔、Ｔ…粘着テープ、Ｔａ…粘着面。 DESCRIPTION OF SYMBOLS 1 ... Sample stand, 2, 2A, 2B, 2C ... Sample support body, 3 ... Detector, 4 ... Laser irradiation part, 10 ... Sample, 11 ... Sample ion, 21 ... Substrate, 21a ... One side, 21b ... Other side, 22, 122 ... Frame, 23 ... Conductive layer, 24 ... Adhesive layer, 24a, 24b ... Adhesive surface, 30 ... Screw, 122a ... Insertion hole, L ... Laser, S ... Through hole, T ... Adhesive tape, Ta ... Adhesive surface.

Claims (10)

  1. 　一面から他面にかけて貫通する複数の貫通孔が設けられた基板と、導電性材料からなり、少なくとも前記一面を覆う導電層と、を備える試料支持体が用意される第１工程と、
    　試料が試料台に載置され、且つ、前記他面が前記試料に対向するように前記試料支持体が前記試料上に配置される第２工程と、
    　前記一面にレーザが照射されることにより、毛細管現象によって前記他面側から前記貫通孔を介して前記一面側に移動した前記試料がイオン化される第３工程と、
    を含む表面支援レーザ脱離イオン化法。     A first step in which a sample support including a substrate provided with a plurality of through holes penetrating from one surface to the other surface and a conductive layer made of a conductive material and covering at least the one surface is prepared;
    A second step in which the sample is placed on the sample stage and the sample support is disposed on the sample such that the other surface faces the sample;
    A third step of ionizing the sample moved from the other surface side to the one surface side through the through hole by capillary action by irradiating the one surface with a laser;
    Surface assisted laser desorption ionization method including:
  2. 　前記基板はバルブ金属又はシリコンを陽極酸化することにより形成されている、
    請求項１に記載の表面支援レーザ脱離イオン化法。     The substrate is formed by anodizing a valve metal or silicon,
    The surface-assisted laser desorption / ionization method according to claim 1.
  3. 　前記貫通孔の幅は１～７００ｎｍである、
    請求項１又は２に記載の表面支援レーザ脱離イオン化法。     The width of the through hole is 1 to 700 nm.
    The surface-assisted laser desorption / ionization method according to claim 1 or 2.
  4. 　前記基板の厚さは５～１０μｍである、
    請求項１～３のいずれか一項に記載の表面支援レーザ脱離イオン化法。     The thickness of the substrate is 5 to 10 μm.
    The surface-assisted laser desorption / ionization method according to any one of claims 1 to 3.
  5. 　前記試料支持体が、前記基板の前記一面の外縁部に取り付けられた枠体を更に備える、
    請求項１～４のいずれか一項に記載の表面支援レーザ脱離イオン化法。     The sample support further comprises a frame attached to an outer edge of the one surface of the substrate;
    The surface-assisted laser desorption / ionization method according to any one of claims 1 to 4.
  6. 　前記第２工程では、前記試料支持体が前記試料台に固定される、
    請求項１～５のいずれか一項に記載の表面支援レーザ脱離イオン化法。     In the second step, the sample support is fixed to the sample stage.
    The surface-assisted laser desorption / ionization method according to any one of claims 1 to 5.
  7. 　導電性材料からなり、一面から他面にかけて貫通する複数の貫通孔が設けられた基板を備える試料支持体が用意される第１工程と、
    　試料が試料台に載置され、且つ、前記他面が前記試料と接触するように前記試料支持体が前記試料上に配置される第２工程と、
    　前記一面にレーザが照射されることにより、毛細管現象によって前記他面側から前記貫通孔を介して前記一面側に移動した前記試料がイオン化される第３工程と、
    を含む表面支援レーザ脱離イオン化法。     A first step of preparing a sample support including a substrate made of a conductive material and provided with a plurality of through holes penetrating from one surface to the other surface;
    A second step in which the sample is placed on the sample stage and the sample support is placed on the sample such that the other surface is in contact with the sample;
    A third step of ionizing the sample moved from the other surface side to the one surface side through the through hole by capillary action by irradiating the one surface with a laser;
    Surface assisted laser desorption ionization method including:
  8. 　請求項１～７のいずれか一項に記載の表面支援レーザ脱離イオン化法の各工程と、
    　前記第３工程においてイオン化された前記試料が検出される第４工程と、
    を含み、
    　前記第３工程での前記レーザの照射、及び前記第４工程でのイオン化された前記試料の検出は、前記レーザの照射位置を変えながら当該照射位置毎に行われる、
    質量分析方法。     Each step of the surface-assisted laser desorption / ionization method according to any one of claims 1 to 7,
    A fourth step in which the sample ionized in the third step is detected;
    Including
    The irradiation of the laser in the third step and the detection of the ionized sample in the fourth step are performed for each irradiation position while changing the irradiation position of the laser.
    Mass spectrometry method.
  9. 　試料が載置される試料台と、
    　一面から他面にかけて貫通する複数の貫通孔が設けられた基板と、導電性材料からなり、少なくとも前記一面を覆う導電層と、を備える試料支持体が、前記他面が前記試料に対向するように、前記試料台に載置された前記試料上に配置された状態で、照射位置を変えながら前記一面にレーザを照射するレーザ照射部と、
    　前記レーザの照射によりイオン化された前記試料を前記照射位置毎に検出する検出部と、
    を備える、質量分析装置。     A sample stage on which the sample is placed;
    A sample support comprising: a substrate provided with a plurality of through holes penetrating from one surface to the other surface; and a conductive layer made of a conductive material and covering at least the one surface so that the other surface faces the sample. In addition, a laser irradiation unit that irradiates the one surface with a laser while changing an irradiation position in a state of being placed on the sample placed on the sample stage,
    A detection unit for detecting the sample ionized by the laser irradiation for each irradiation position;
    A mass spectrometer.
  10. 　試料が載置される試料台と、
    　導電性材料からなり、一面から他面にかけて貫通する複数の貫通孔が設けられた基板を備える試料支持体が、前記他面が前記試料と接触するように、前記試料台に載置された前記試料上に配置された状態で、照射位置を変えながら前記一面にレーザを照射するレーザ照射部と、
    　前記レーザの照射によりイオン化された前記試料を前記照射位置毎に検出する検出部と、
    を備える、質量分析装置。     A sample stage on which the sample is placed;
    A sample support comprising a substrate made of a conductive material and provided with a plurality of through holes penetrating from one surface to the other surface is placed on the sample stage so that the other surface is in contact with the sample. A laser irradiation unit that irradiates a laser on the one surface while changing the irradiation position in a state of being arranged on the sample;
    A detection unit for detecting the sample ionized by the laser irradiation for each irradiation position;
    A mass spectrometer.
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